Identification of Tumor-Associated Antigens for Diagnosis and Therapy

ABSTRACT

The invention relates to genetic products the expression of which is associated with cancer diseases. The invention also relates to the therapy and diagnosis of diseases in which the genetic products are expressed or aberrantly expressed, in particular cancer diseases.

Despite interdisciplinary approaches and exhaustive use of classicaltherapeutic procedures, cancers are still among the leading causes ofdeath. More recent therapeutic concepts aim at incorporating thepatient's immune system into the overall therapeutic concept by usingrecombinant tumor vaccines and other specific measures such as antibodytherapy. A prerequisite for the success of such a strategy is therecognition of tumor-specific or tumor-associated antigens or epitopesby the patient's immune system whose effector functions are to beinterventionally enhanced. Tumor cells biologically differ substantiallyfrom their nonmalignant cells of origin. These differences are due togenetic alterations acquired during tumor development and result, interalia, also in the formation of qualitatively or quantitatively alteredmolecular structures in the cancer cells. Tumor-associated structures ofthis kind which are recognized by the specific immune system of thetumor-harboring host are referred to as tumor-associated antigens. Thespecific recognition of tumor-associated antigens involves cellular andhumoral mechanisms which are two functionally interconnected units: CD4⁺and CD8⁺ T lymphocytes recognize the processed antigens presented on themolecules of the MHC (major histocompatibility complex) classes II andI, respectively, while B lymphocytes produce circulating antibodymolecules which bind directly to unprocessed antigens. The potentialclinical-therapeutical importance of tumor-associated antigens resultsfrom the fact that the recognition of antigens on neoplastic cells bythe immune system leads to the initiation of cytotoxic effectormechanisms and, in the presence of T helper cells, can cause eliminationof the cancer cells (Pardoll, Nat. Med. 4:525-31, 1998). Accordingly, acentral aim of tumor immunology is to molecularly define thesestructures. The molecular nature of these antigens has been enigmaticfor a long time. Only after development of appropriate cloningtechniques has it been possible to screen cDNA expression libraries oftumors systematically for tumor-associated antigens by analyzing thetarget structures of cytotoxic T lymphocytes (CTL) (van der Bruggen etal., Science 254:1643-7, 1991) or by using circulating autoantibodies(Sahin et al., Curr. Opin. Immunol. 9:709-16, 1997) as probes. To thisend, cDNA expression libraries were prepared from fresh tumor tissue andrecombinantly expressed as proteins in suitable systems. Immunoeffectorsisolated from patients, namely CTL clones with tumor-specific lysispatterns, or circulating autoantibodies were utilized for cloning therespective antigens.

In recent years a multiplicity of antigens have been defined in variousneoplasias by these approaches.

However, the probes utilized for antigen identification in the classicalmethods are immunoeffectors (circulating autoantibodies or CTL clones)from patients usually having already advanced cancer. A number of dataindicate that tumors can lead, for example, to tolerization andanergization of T cells and that, during the course of the disease,especially those specificities which could cause effective immunerecognition are lost from the immunoeffector repertoire. Current patientstudies have not yet produced any solid evidence of a real action of thepreviously found and utilized tumor-associated antigens. Accordingly, itcannot be ruled out that proteins evoking spontaneous immune responsesare the wrong target structures.

It was the object of the present invention to provide target structuresfor a diagnosis and therapy of cancers.

This object is achieved by the subject matter of the claims.

According to the invention, genes are identified which are selectivelyor aberrantly expressed in tumor cells and thus, providetumor-associated antigens. These genes and/or their genetic productsand/or the derivatives and/or fragments thereof are useful as targetstructures for therapeutic and diagnostic approaches.

The tumor-associated antigens identified according to the invention havean amino acid sequence encoded by a nucleic acid which is selected fromthe group consisting of (a) a nucleic acid which comprises a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9,13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67 of thesequence listing, a part or derivative thereof, (b) a nucleic acid whichhybridizes with the nucleic acid of (a) under stringent conditions, (c)a nucleic acid which is degenerate with respect to the nucleic acid of(a) or (b), and (d) a nucleic acid which is complementary to the nucleicacid of (a), (b) or (c). In a preferred embodiment, a tumor-associatedantigen identified according to the invention has an amino acid sequenceencoded by a nucleic acid which is selected from the group consisting ofSEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62,and 64-67 of the sequence listing. In a further preferred embodiment, atumor-associated antigen identified according to the invention comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:2, 6, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69of the sequence listing, a part or derivative thereof.

The present invention generally relates to the use of tumor-associatedantigens identified according to the invention or of parts orderivatives thereof, of nucleic acids coding for the tumor-associatedantigens identified according to the invention or of parts orderivatives thereof or of nucleic acids directed against said codingnucleic acids, of antibodies or T cells directed against thetumor-associated antigens identified according to the invention or partsor derivatives thereof and/or of host cells expressing thetumor-associated antigens identified according to the invention or partsor derivatives thereof for therapy, prophylaxis, diagnosis and/ormonitoring of neoplastic diseases. This may also involve the use of acombination of two or more of these antigens, nucleic acids, antibodies,T cells and/or host cells, in one embodiment also in combination withtumor-associated antigens other than those identified according to theinvention, nucleic acids coding therefor or nucleic acids directedagainst said coding nucleic acids, antibodies or T cells directedagainst said tumor-associated antigens and/or host cells expressing saidtumor associated antigens.

In those embodiments of the invention relating to the use of antibodiesdirected against the tumor-associated antigens identified according tothe invention or parts or derivatives thereof also T cell receptorsdirected against the tumor-associated antigens identified according tothe invention or parts or derivatives thereof, optionally in a complexwith MHC molecules, may be used.

Especially suitable for therapy, prophylaxis, diagnosis and/ormonitoring is a part of the tumor-associated antigens identifiedaccording to the invention which corresponds to the non-transmembraneportion, in particular the extracellular portion of the tumor-associatedantigens or is comprised thereof. Therefore, according to the invention,a part of the tumor-associated antigens identified according to theinvention which corresponds to the non-transmembrane portion, inparticular the extracellular portion of the tumor-associated antigens oris comprised thereof, or a corresponding part of the nucleic acidscoding for the tumor-associated antigens identified according to theinvention is preferred for therapy, prophylaxis, diagnosis and/ormonitoring. Similarly the use of antibodies is preferred which aredirected against a part of the tumor-associated antigens identifiedaccording to the invention which corresponds to the non-transmembraneportion, in particular the extracellular portion of the tumor-associatedantigens or is comprised thereof.

Preferred diseases for a therapy, prophylaxis and/or diagnosis are thosein which one or more of the tumor-associated antigens identifiedaccording to the invention are selectively expressed or abnormallyexpressed.

Furthermore, the invention relates to nucleic acids and proteins orpeptides, which result from altered splicing (splice variants) of knowngenes or altered translation using alternative open reading frames. Inthis aspect the invention relates to nucleic acids which comprise anucleic acid sequence selected from the group consisting of SEQ ID NOs:28 and 49 of the sequence listing. Moreover, in this aspect, theinvetion relates to proteins or peptides which comprise an amino acidsequence selected from the group consisting of SEQ ID NOs: 29 and 50 ofthe sequence listing.

Altered splicing of a gene results in an altered transcript sequence(splice variant). Translation of a splice variant in the region of itsaltered sequence results in an altered protein which may be distinctlydifferent in the structure and function from the original protein.Tumor-associated splice variants may produce tumor-associatedtranscripts and tumor-associated proteins/antigens. These may beutilized as molecular markers both for detecting tumor cells and fortherapeutic targeting of tumors. Detection of tumor cells in a samplefrom a patient may be carried out according to the invention, forexample, after extraction of nucleic acids by PCR amplification withsplice variant-specific oligonucleotides.

According to the invention, all sequence-dependent detection systems aresuitable for detection. These are, apart from PCR, for example genechip/microarray systems, Northern blot, RNAse protection assays (RDA)and others. All detection systems have in common that detection is basedon a specific hybridization with at least one splice variant-specificnucleic acid sequence. However, tumor cells may also be detectedaccording to the invention by antibodies which recognize a specificepitope encoded by the splice variant. Said antibodies may be preparedby using for immunization peptides which are specific for said splicevariant. Suitable for immunization are particularly the amino acidsequences which are distinctly different from the variant(s) of thegenetic product, which is (are) preferably produced in healthy cells.Detection of the tumor cells with antibodies may be carried out here ona sample isolated from the patient or as imaging with intravenouslyadministered antibodies.

In addition to diagnostic usability, splice variants having new oraltered epitopes are attractive targets for immunotherapy as theseepitopes may be utilized for targeting antibodies or T lymphocytes asdescribed herein. In passive immunotherapy, antibodies or T lymphocyteswhich recognize splice variant-specific epitopes are adoptivelytransferred here. As in the case of other antigens, antibodies may begenerated also by using standard technologies with utilization ofpolypeptides which include these epitopes. Alternatively, it is possibleto utilize for immunization nucleic acids coding for peptides whichcontain said epitopes. Various techniques for in vitro or in vivogeneration of epitope-specific T lymphocytes are known and have beendescribed in detail (for example Kessler J H, et al. 2001, Sahin et al.,1997) and are likewise based on utilizing peptides which contain thesplice variant-specific epitopes or nucleic acids coding for saidpeptides. Peptides which contain the splice variant-specific epitopes ornucleic acids coding for said peptides may also be used aspharmaceutically active substances in active immunotherapy (e.g.vaccination, vaccine therapy).

In one aspect, the invention relates to a pharmaceutical compositioncomprising an agent which recognizes a tumor-associated antigenidentified according to the invention or a nucleic acid coding for thetumor-associated antigen and which is preferably selective for cellswhich have expression or abnormal expression of a tumor-associatedantigen identified according to the invention. In a further aspect, theinvention relates to a pharmaceutical composition comprising an agentwhich (I) inhibits expression or activity of a tumor-associated antigenidentified according to the invention, and/or (II) has tumor-inhibitingor tumor-destroying activity and is selective for cells expressing orabnormally expressing a tumor-associated antigen identified according tothe invention, and/or (III) when administered, selectively increases theamount of complexes between an MHC molecule and a tumor-associatedantigen identified according to the invention or a part thereof, such asa peptide epitope. In particular embodiments, said agent may causeinduction of cell death, reduction in cell growth, damage to the cellmembrane or secretion of cytokines and preferably have atumor-inhibiting activity. In one embodiment, the agent is an antisensenucleic acid which hybridizes selectively with the nucleic acid codingfor the tumor-associated antigen. In a further embodiment, the agent isa siRNA preferably comprising a sense RNA strand and an antisense RNAstrand, wherein the sense and antisense RNA strands form an RNA duplex,and wherein the sense RNA strand comprises a nucleotide sequencesubstantially identical to a target sequence of about 19 to about 25contiguous nucleotides in a nucleic acid coding for the tumor-associatedantigen, preferably mRNA coding for the tumor-associated antigen. In afurther embodiment, the agent is an antibody which binds selectively tothe tumor-associated antigen, in particular a complement-activated ortoxin conjugated antibody which binds selectively to thetumor-associated antigen. In a preferred embodiment, the antibody whichbinds selectively to the tumor-associated antigen is coupled to atherapeutically useful substance and/or recruits natural or artificialeffector mechanisms to said cell expressing or abnormally expressingsaid tumor-associated antigen. In a further embodiment, the agent is acytotoxic T lymphocyte which recognizes the tumor-associated antigen ora part thereof bound by an MHC molecule on a cell and lyses the cellslabeled in this way. In a further embodiment, the agent is a T helperlymphocyte which enhances effector functions of other cells specificallyrecognizing said tumor-associated antigen or a part thereof.

In a further embodiment, the agent comprises two or more agents whicheach recognize different tumor-associated antigens and/or inhibitexpression or activity of different tumor-associated antigens, and/orhave tumor-inhibiting or tumor-destroying activity and are selective forcells expressing or abnormally expressing different tumor-associatedantigens, and/or when administered, selectively increase the amount ofcomplexes between MHC molecules and different tumor-associated antigensor parts thereof, wherein at least one of said differenttumor-associated antigens is a tumor-associated antigen identifiedaccording to the invention. Preferably, a tumor-associated antigenselectively limited to tumors serves as a label for recruiting effectormechanisms to this specific location. The invention includes embodimentswherein the agent itself does not have an ability to inhibit activity ofa tumor-associated antigen or a tumor-inhibiting or tumor-destroyingactivity but mediates such effect, in particular by recruiting effectormechanisms, in particular those having cell damaging potential, to aspecific location, in particular a tumor or tumor cells.

The activity of a tumor-associated antigen identified according to theinvention can be any activity of a protein or a peptide. In oneembodiment this activity is an enzymatic activity.

According to the invention the phrase “inhibit expression or activity”includes a complete or essentially complete inhibition of expression oractivity and a reduction in expression or activity.

The agent which, when administered, selectively increases the amount ofcomplexes between an MHC molecule and a tumor-associated antigenidentified according to the invention or a part thereof comprises one ormore components selected from the group consisting of (i) thetumor-associated antigen or a part thereof, (ii) a nucleic acid whichcodes for said tumor-associated antigen or a part thereof, (iii) a hostcell which expresses said tumor-associated antigen or a part thereof,and (iv) isolated complexes between peptide epitopes from saidtumor-associated antigen and an MHC molecule.

The invention furthermore relates to a pharmaceutical composition whichcomprises one or more components selected from the group consisting of(i) a tumor-associated antigen identified according to the invention ora part thereof, (ii) a nucleic acid which codes for a tumor-associatedantigen identified according to the invention or a part thereof, (iii)an antibody which binds to a tumor-associated antigen identifiedaccording to the invention or to a part thereof, (iv) an antisensenucleic acid which hybridizes specifically with a nucleic acid codingfor a tumor-associated antigen identified according to the invention,(v) an siRNA directed against a nucleic acid coding for atumor-associated antigen identified according to the invention, (vi) ahost cell which expresses a tumor-associated antigen identifiedaccording to the invention or a part thereof, and (vii) isolatedcomplexes between a tumor-associated antigen identified according to theinvention or a part thereof and an MHC molecule.

In one embodiment, a nucleic acid coding for a tumor-associated antigenidentified according to the invention or a part thereof is present inthe pharmaceutical composition in an expression vector and functionallylinked to a promoter. In a further embodiment, a nucleic acid coding fora tumor-associated antigen identified according to the invention or apart thereof is present in the pharmaceutical composition in a virus asfurther described below.

In a further embodiment, a host cell present in a pharmaceuticalcomposition of the invention secretes the tumor-associated antigen orthe part thereof, expresses it on the surface and preferablyadditionally express an MHC molecule which binds to saidtumor-associated antigen or said part thereof. In one embodiment, thehost cell expresses the MHC molecule endogenously. In a furtherembodiment, the host cell expresses the MHC molecule and/or thetumor-associated antigen or the part thereof in a recombinant manner.The host cell is preferably nonproliferative. In a preferred embodiment,the host cell is an antigen-presenting cell, in particular a dendriticcell, a monocyte or a macrophage.

In a further embodiment, an antibody present in a pharmaceuticalcomposition of the invention is a monoclonal antibody. In furtherembodiments, the antibody is a chimeric or humanized antibody, afragment of a natural antibody or a synthetic antibody. The antibody maybe coupled to a therapeutically or diagnostically useful agent alsotermed therapeutic or diagnostic agent herein.

An antisense nucleic acid present in a pharmaceutical composition of theinvention may comprise a sequence of 6-50, in particular 10-30, 15-30and 20-30, contiguous nucleotides of the nucleic acid coding for thetumor-associated antigen identified according to the invention.

In further embodiments, a tumor-associated antigen or a part thereof,provided by a pharmaceutical composition of the invention eitherdirectly or via expression of a nucleic acid, binds to MHC molecules onthe surface of cells, said binding preferably causing a cytolyticresponse and/or inducing cytokine release.

In particular embodiments of the siRNA targeting the nucleic acidaccording to SEQ ID NO: 1 the sense RNA strand has the sequence of SEQID NO: 70 and the antisense RNA strand has the sequence of SEQ ID NO:71, or the sense RNA strand has the sequence of SEQ ID NO: 72 and theantisense RNA strand has the sequence of SEQ ID NO: 73.

A pharmaceutical composition of the invention may comprise apharmaceutically compatible carrier and/or an adjuvant.

A pharmaceutical composition of the invention is preferably used for thetreatment or prevention of a disease characterized by selectiveexpression or abnormal expression of a tumor-associated antigen. In apreferred embodiment, the disease is a neoplastic disease, preferablycancer.

In a preferred embodiment, the pharmaceutical composition of theinvention is in the form of a vaccine which may be used therapeuticallyor prophylactically. Such vaccine preferably comprises atumor-associated antigen identified according to the invention or a partthereof, and/or a nucleic acid which codes for a tumor-associatedantigen identified according to the invention or a part thereof. Inparticular embodiments, the nucleic acid is present in a virus or hostcell.

The invention furthermore relates to methods of treating, preventing,diagnosing or monitoring, i.e. determining the regression, progression,course and/or onset of, a disease characterized by expression orabnormal expression of one of more tumor-associated antigens identifiedaccording to the invention, preferably a neoplastic disease, inparticular cancer. In one embodiment, the treatment or preventioncomprises administering a pharmaceutical composition of the invention.

Said methods of diagnosing and/or methods of monitoring according to theinvention generally concern the detection of and/or determination of thequantity of one or more parameters selected from the group consisting of(i) a nucleic acid, which codes for a tumor-associated antigenidentified according to the invention, or a part thereof, (ii) atumor-associated antigen identified according to the invention, or apart thereof (iii) an antibody against a tumor-associated antigenidentified according to the invention or a part thereof, and (iv) Tlymphocytes, preferably cytotoxic or T helper lymphocytes, which arespecific for a tumor-associated antigen identified according to theinvention or a part thereof and/or a complex between thetumor-associated antigen or a part thereof and an MHC molecule, in abiological sample isolated from a patient, preferably from a patienthaving said disease, being suspected of having or falling ill with saiddisease or having a potential for said disease. Means for accomplishingsaid detection and/or determination of the quantity are described hereinand will be apparent to the skilled person.

Preferably, the presence of said nucleic acid, said tumor-associatedantigen or said part thereof, said antibody and/or said T lymphocytesand/or a quantity of said nucleic acid, said tumor-associated antigen orsaid part thereof, said antibody and/or said T lymphocytes which isincreased compared to a patient without said disease is indicative forthe presence of said disease or a potential for a development of saiddisease.

The methods of diagnosing and/or monitoring of the invention alsoinclude embodiments wherein by detection or determination of thequantity of said nucleic acid, said tumor-associated antigen or saidpart thereof, said antibody and/or said T lymphocytes it is possible toassess and/or prognose the metastatic behavior of said disease, wherein,preferably, the presence of said nucleic acid, said tumor-associatedantigen or said part thereof, said antibody and/or said T lymphocytesand/or a quantity of said nucleic acid, said tumor-associated antigen orsaid part thereof, said antibody and/or said T lymphocytes which isincreased compared to a patient without said disease or without ametastasis of said disease is indicative for a metastatic behavior ofsaid disease or a potential for a metastatic behavior of said disease.

In particular embodiments, said detection or determination of thequantity comprises (i) contacting a biological sample with an agentwhich binds specifically to said nucleic acid coding for thetumor-associated antigen or said part thereof, to said tumor-associatedantigen or said part thereof, to said antibody or said part thereof orto said T lymphocytes, and (ii) detecting the formation of ordetermining the amount of a complex between the agent and the nucleicacid or the part thereof, the tumor-associated antigen or the partthereof, the antibody or the part thereof, or the T lymphocytes. In oneembodiment, the disease is characterized by expression or abnormalexpression of two or more different tumor-associated antigens and adetection or determination of the amount comprises a detection ordetermination of the amount of two or more nucleic acids coding for saidtwo or more different tumor-associated antigens or of parts thereof, oftwo or more different tumor-associated antigens or of parts thereof, oftwo or more antibodies binding to said two or more differenttumor-associated antigens or to parts thereof and/or of two or more Tlymphocytes specific for said two or more different tumor-associatedantigens or parts thereof, or complexes thereof with MHC molecules. In afurther embodiment, the biological sample isolated from the patient iscompared to a comparable normal biological sample.

The methods of monitoring according to the invention preferably comprisea detection of and/or determination of the quantity of one or more ofthe parameters mentioned above in a first sample at a first point intime and in a further sample at a second point in time, wherein thecourse of the disease is determined by comparing the two samples.

According to the invention, detection of a nucleic acid or of a partthereof or determining the quantity of a nucleic acid or of a partthereof may be carried out using a oligo- or polynucleotide probe whichhybridizes specifically to said nucleic acid or said part thereof or maybe carried out by selective amplification of said nucleic acid or saidpart thereof, e.g. by means of PCR amplification. In one embodiment, theoligo- or polynucleotide probe comprises a sequence of 6-50, inparticular 10-30, 15-30 and 20-30, contiguous nucleotides of saidnucleic acid.

In particular embodiments, the tumor-associated antigen or the partthereof which is to be detected or the amount of which is to bedetermined in the methods of the present invention is presentintracellularly, on the cell surface or in a complex with an MHCmolecule. According to the invention, detection of a tumor-associatedantigen or of a part thereof or determining the quantity of atumor-associated antigen or of a part thereof may be carried out usingan antibody binding specifically to said tumor-associated antigen orsaid part thereof.

According to the invention, detection of an antibody or determining thequantity of an antibody may be carried out using a protein or peptidebinding specifically to said antibody.

According to the invention, detection of or determining the quantity ofT lymphocytes which are specific for a tumor-associated antigen or apart thereof and/or a complex thereof with an MHC molecule may becarried out using a cell presenting the complex between saidtumor-associated antigen or said part thereof and an MHC molecule. Tlymphocytes may additionally be detected by detecting theirproliferation, their cytokine production, and their cytotoxic activitytriggered by specific stimulation with a complex of an MHC molecule anda tumor-associated antigen or a part thereof. T lymphocytes may also bedetected with aid of a recombinant MHC molecule or a complex of two ormore MHC molecules loaded with immunogenic fragments of one or moretumor-associated antigens.

An agent which is used for detection or determining the quantity in themethods of the invention such as a oligo- or polynucleotide probe, anantibody, a protein or peptide or a cell is preferably labeled in adetectable manner, in particular by a detectable marker such as aradioactive marker or an enzymic marker.

In a particular aspect, the invention relates to a method of treating,preventing, diagnosing or monitoring a disease characterized byexpression or abnormal expression of a tumor-associated antigenidentified according to the invention, which method comprisesadministering an antibody which binds to said tumor-associated antigenor to a part thereof and which is coupled to a therapeutic or diagnosticagent. The antibody may be a monoclonal antibody. In furtherembodiments, the antibody is a chimeric or humanized antibody or afragment of a natural antibody.

In certain embodiments, the methods of the invention of diagnosing ormonitoring a disease characterized by expression or abnormal expressionof a tumor-associated antigen identified according to the invention areperformed with a biological sample containing or suspected of containingdisseminating tumor cells or metastatic tumor cells. Such biologicalsamples include, for example, blood, serum, bone marrow, sputum,bronchial aspirate, and/or bronchial lavage.

In one particular aspect, the invention relates to a method of treatinga patient having a disease characterized by expression or abnormalexpression of a tumor-associated antigen identified according to theinvention, which method comprises (i) providing a sample containingimmunoreactive cells, either obtained from said patient or from anotherindividual of the same species, in particular a healthy individual, oran individual of a different species, (ii) contacting said sample with ahost cell expressing said tumor-associated antigen or a part thereof,under conditions which favor production of cytolytic T cells againstsaid tumor-associated antigen or a part thereof, and (iii) introducingthe cytolytic T cells into the patient in an amount suitable for lysingcells expressing the tumor-associated antigen or a part thereof. In oneembodiment, the method includes cloning of the T cell receptor ofcytolytic T cells obtained and transferring the nucleic acid coding forthe T cell receptor to T cells, either obtained from said patient orfrom another individual of the same species, in particular a healthyindividual, or an individual of a different species, which T cells thusreceive the desired specificity and, as under (iii), may be introducedinto the patient.

In one embodiment, the host cell endogenously expresses an MHC molecule.In a further embodiment, the host cell recombinantly expresses an MHCmolecule and/or the tumor-associated antigen or the part thereof.Preferably, the host cell presents the tumor-associated antigen or thepart thereof by MHC molecules on its surface. The host cell ispreferably nonproliferative. In a preferred embodiment, the host cell isan antigen-presenting cell, in particular a dendritic cell, a monocyteor a macrophage.

The invention also relates to a method of treating a diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen identified according to the invention, which method comprises(i) identifying cells from the patient which express abnormal amounts ofthe tumor-associated antigen, (ii) isolating a sample of said cells,(iii) culturing said cells, and (iv) introducing said cells into thepatient in an amount suitable for triggering an immune response to thecells.

The present invention furthermore relates to a nucleic acid selectedfrom the group consisting of (a) a nucleic acid which comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67,a part or derivative thereof, (b) a nucleic acid which hybridizes withthe nucleic acid of (a) under stringent conditions, (c) a nucleic acidwhich is degenerate with respect to the nucleic acid of (a) or (b), and(d) a nucleic acid which is complementary to the nucleic acid of (a),(b) or (c). The invention furthermore relates to a nucleic acid, whichcodes for a protein or polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22,26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69, a part or derivativethereof.

In a further aspect, the invention relates to a recombinant nucleic acidmolecule, in particular DNA or RNA molecule, which comprises a nucleicacid of the invention.

The invention also relates to host cells which contain a nucleic acid orrecombinant nucleic acid molecule of the invention.

The host cell may also comprise a nucleic acid coding for a MHCmolecule. In one embodiment, the host cell endogenously expresses theMHC molecule. In a further embodiment, the host cell recombinantlyexpresses the MHC molecule and/or the nucleic acid or recombinantnucleic acid molecule of the invention or a part thereof. Preferably,the host cell is nonproliferative. In a preferred embodiment, the hostcell is an antigen-presenting cell, in particular a dendritic cell, amonocyte or a macrophage.

In a further embodiment, the invention relates to oligonucleotides whichhybridize with a nucleic acid identified according to the invention andwhich may be used as genetic probes or as “antisense” molecules. Nucleicacid molecules in the form of oligonucleotide primers or competentprobes, which hybridize with a nucleic acid identified according to theinvention or parts thereof, may be used for finding nucleic acids whichare homologous to said nucleic acid identified according to theinvention, e.g. by PCR amplification, Southern and Northernhybridization. Hybridization may be carried out under low stringency,more preferably under medium stringency and most preferably under highstringency conditions.

In a further aspect, the invention relates to a protein or peptide whichis encoded by a nucleic acid selected from the group consisting of (a) anucleic acid which comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39,41, 45, 49, 61, 62, and 64-67, a part or derivative thereof, (b) anucleic acid which hybridizes with the nucleic acid of (a) understringent conditions, (c) a nucleic acid which is degenerate withrespect to the nucleic acid of (a) or (b), and (d) a nucleic acid whichis complementary to the nucleic acid of (a), (b) or (c). In a preferredembodiment, the invention relates to a protein or peptide whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63,68, and 69, a part or derivative thereof.

In a further aspect, the invention relates to an immunogenic fragment ofa tumor-associated antigen identified according to the invention. Saidfragment preferably binds to a MHC molecule or an antibody, preferablyto a human HLA receptor or a human antibody. According to the invention,a fragment preferably comprises a sequence of at least 6, in particularat least 8, at least 10, at least 12, at least 15, at least 20, at least30 or at least 50, amino acids.

In this aspect the invention relates, in particular, to a peptide whichhas or comprises a sequence selected from the group consisting of SEQ IDNOs: 51-60, 68 and of the sequence listing, a part or derivativethereof.

In a further aspect, the invention relates to an agent which binds to atumor-associated antigen identified according to the invention or to apart thereof. In a preferred embodiment, the agent is a protein orpeptide, in particular an antibody, a T cell receptor or an MHCmolecule. In further embodiments, the antibody is a monoclonal,chimeric, or humanized antibody, an antibody produced by combinatorytechniques, or a fragment of an antibody. In one preferred embodiment,the invention relates to an antibody which binds selectively to acomplex of (i) a tumor-associated antigen identified according to theinvention or a part thereof and (ii) an MHC molecule to which saidtumor-associated antigen identified according to the invention or saidpart thereof binds, with said antibody not binding to (i) or (ii) alone.

In particular, the invention relates to such an agent, in particular anantibody, which specifically binds to a peptide which has or comprises asequence selected from the group consisting of SEQ ID NOs: 51-60, 68,and of the sequence listing, a part or derivative thereof.

According to the invention, the term “binding” preferably relates to aspecific binding. “Specific binding” means that an agent such as anantibody binds stronger to a target such as an epitope for which it isspecific compared to the binding to another target. An agent bindsstronger to a first target compared to a second target if it binds tothe first target with a dissociation constant (K_(D)) which is lowerthan the dissociation constant for the second target. Preferably thedissociation constant (K_(D)) for the target to which the agent bindsspecifically is more than 10-fold, preferably more than 20-fold, morepreferably more than 50-fold, even more preferably more than 100-fold,200-fold, 500-fold or 1000-fold lower than the dissociation constant(K_(D)) for the target to which the agent does not bind specifically.

Such specific antibodies may, for example, be obtained by immunizationusing the aforementioned peptides.

The invention furthermore relates to a conjugate between an agent of theinvention which binds to a tumor-associated antigen identified accordingto the invention or to a part thereof or an antibody of the inventionand a therapeutic or diagnostic agent. In one embodiment, thetherapeutic or diagnostic agent is a toxin.

In a further aspect, the invention relates to a kit for detectingexpression or abnormal expression of a tumor-associated antigenidentified according to the invention, which kit comprises agents fordetection or determining the quantity (i) of the nucleic acid whichcodes for the tumor-associated antigen or of a part thereof, (ii) of thetumor-associated antigen or of a part thereof, (iii) of antibodies whichbind to the tumor-associated antigen or to a part thereof, and/or (iv)of T cells which are specific for the tumor-associated antigen or a partthereof or a complex thereof with an MHC molecule. In one embodiment,the agents for detection of the nucleic acid or the part thereof arenucleic acid molecules for selective amplification of said nucleic acid,which comprise, in particular, a sequence of 6-50, in particular 10-30,15-30 and 20-30, contiguous nucleotides of said nucleic acid.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, a “reference” such as a reference sample orreference organism may be used to correlate and compare the resultsobtained in the methods of the invention from a test sample or testorganism, i.e. a patient. Typically the reference organism is a healthyorganism, in particular an organism which does not suffer from cancer.

A “reference value” can be determined from a reference empirically bymeasuring a sufficiently large number of references. Preferably thereference value is determined by measuring at least 2, preferably atleast 3, preferably at least 5, preferably at least 8, preferably atleast 12, preferably at least 20, preferably at least 30, preferably atleast 50, or preferably at least 100 references.

“Derivative” of a nucleic acid means according to the invention thatsingle or multiple such as at least 2, at least 4, or at least 6 andpreferably up to 3, up to 4, up to 5, up to 6, up to 10, up to 15, or upto 20 nucleotide substitutions, deletions and/or additions are presentin said nucleic acid. Furthermore, the term “derivative” also compriseschemical derivatization of a nucleic acid on a nucleotide base, on thesugar or on the phosphate. The term “derivative” also comprises nucleicacids which contain nucleotides and nucleotide analogs not occurringnaturally.

According to the invention, a nucleic acid is preferablydeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acidscomprise according to the invention genomic DNA, cDNA, mRNA,recombinantly produced and chemically synthesized molecules. Accordingto the invention, a nucleic acid may be present as a single-stranded ordouble-stranded and linear or covalently circularly closed molecule.

As used herein, the term “RNA” means a molecule comprising at least oneribonucleotide residue. By “ribonucleotide” is meant a nucleotide with ahydroxyl group at the 2′-position of a beta-D-ribo-furanose moiety. Theterm includes double stranded RNA, single stranded RNA, isolated RNAsuch as partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of a RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in RNA molecules can also comprise non-standard nucleotides,such as non-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs or analogs of naturally-occurring RNA.

The nucleic acids described according to the invention have preferablybeen isolated. The term “isolated nucleic acid” means according to theinvention that the nucleic acid was (i) amplified in vitro, for exampleby polymerase chain reaction (PCR), (ii) recombinantly produced bycloning, (iii) purified, for example by cleavage and gel-electrophoreticfractionation, or (iv) synthesized, for example by chemical synthesis.An isolated nucleic acid is a nucleic acid which is available formanipulation by recombinant DNA techniques.

A nucleic acid is “complementary” to another nucleic acid if the twosequences are capable of hybridizing and forming a stable duplex withone another, with hybridization preferably being carried out underconditions which allow specific hybridization between polynucleotides(stringent conditions). Stringent conditions are described, for example,in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors,2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor,N.Y., 1989 or Current Protocols in Molecular Biology, F. M. Ausubel etal., Editors, John Wiley & Sons, Inc., New York and refer, for example,to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mMNaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15M sodium citrate, pH 7. After hybridization, the membrane to which theDNA has been transferred is washed, for example, in 2×SSC at roomtemperature and then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68°C.

According to the invention, complementary nucleic acids have at least40%, in particular at least 50%, at least 60%, at least 70%, at least80%, at least 90% and preferably at least 95%, at least 98% or at least99%, identical nucleotides.

The term “percentage identity” is intended to denote a percentage ofnucleotides or of amino acid residues which are identical between thetwo sequences to be compared, obtained after the best alignment, thispercentage being purely statistical and the differences between the twosequences being distributed randomly and over their entire length.Sequence comparisons between two nucleotide or amino acid sequences areconventionally carried out by comparing these sequences after havingaligned them optimally, said comparison being carried out by segment orby “window of comparison” in order to identify and compare local regionsof sequence similarity. The optimal alignment of the sequences forcomparison may be produced, besides manually, by means of the localhomology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482,by means of the local homology algorithm of Neddleman and Wunsch, 1970,J. Mol. Biol. 48, 443, by means of the similarity search method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or bymeans of computer programs which use these algorithms (GAP, BESTFIT,FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

Nucleic acids coding for tumor-associated antigens may, according to theinvention, be present alone or in combination with other nucleic acids,in particular heterologous nucleic acids. In preferred embodiments, anucleic acid is functionally linked to expression control sequences orregulatory sequences which may be homologous or heterologous withrespect to said nucleic acid. A coding sequence and a regulatorysequence are “functionally” linked to one another, if they arecovalently linked to one another in such a way that expression ortranscription of said coding sequence is under the control or under theinfluence of said regulatory sequence. If the coding sequence is to betranslated into a functional protein, then, with a regulatory sequencefunctionally linked to said coding sequence, induction of saidregulatory sequence results in transcription of said coding sequence,without causing a frame shift in the coding sequence or said codingsequence not being capable of being translated into the desired proteinor peptide.

The term “expression control sequence” or “regulatory sequence”comprises according to the invention promoters, enhancers and othercontrol elements which regulate expression of a gene. In particularembodiments of the invention, the expression control sequences can beregulated. The exact structure of regulatory sequences may vary as afunction of the species or cell type, but generally comprises 5′untranscribed and 5′ untranslated sequences which are involved ininitiation of transcription and translation, respectively, such as TATAbox, capping sequence, CAAT sequence, and the like. More specifically,5′ untranscribed regulatory sequences comprise a promoter region whichincludes a promoter sequence for transcriptional control of thefunctionally linked gene. Regulatory sequences may also compriseenhancer sequences or upstream activator sequences.

According to the invention, a nucleic acid may furthermore be present incombination with another nucleic acid which codes for a peptidecontrolling secretion of the protein or peptide encoded by said nucleicacid from a host cell. According to the invention, a nucleic acid mayalso be present in combination with another nucleic acid which codes fora peptide causing the encoded protein or peptide to be anchored on thecell membrane of the host cell or compartmentalized into particularorganelles of said cell. Similarly, a combination with a nucleic acid ispossible which represents a reporter gene or any “tag”.

In a preferred embodiment, a recombinant nucleic acid molecule isaccording to the invention a vector, where appropriate with a promoter,which controls expression of a nucleic acid, for example a nucleic acidcoding for a tumor-associated antigen identified according to theinvention. The term “vector” is used here in its most general meaningand comprises any intermediary vehicle for a nucleic acid which enablessaid nucleic acid, for example, to be introduced into prokaryotic and/oreukaryotic cells and, where appropriate, to be integrated into a genome.Vectors of this kind are preferably replicated and/or expressed in thecells. An intermediary vehicle may be adapted, for example, to the usein electroporation, in bombardment with microprojectiles, in liposomaladministration, in the transfer with the aid of agrobacteria or ininsertion via DNA or RNA viruses. Vectors comprise plasmids, phagemids,bacteriophages or viral genomes.

The nucleic acids coding for a tumor-associated antigen identifiedaccording to the invention may be used for transfection of host cells.Nucleic acids here mean both recombinant DNA and RNA. Recombinant RNAmay be prepared by in-vitro transcription of a DNA template.Furthermore, it may be modified by stabilizing sequences, capping andpolyadenylation prior to application.

According to the invention, the term “host cell” relates to any cellwhich can be transformed or transfected with an exogenous nucleic acid.The term “host cells” comprises according to the invention prokaryotic(e.g. E. coli) or eukaryotic cells (e.g. dendritic cells, B cells, CHOcells, COS cells, K562 cells, yeast cells and insect cells). Particularpreference is given to mammalian cells such as cells from humans, mice,hamsters, pigs, goats, primates. The cells may be derived from amultiplicity of tissue types and comprise primary cells and cell lines.Specific examples comprise keratinocytes, peripheral blood leukocytes,stem cells of the bone marrow and embryonic stem cells. In furtherembodiments, the host cell is an antigen-presenting cell, in particulara dendritic cell, monocyte or a macrophage. A nucleic acid may bepresent in the host cell in the form of a single copy or of two or morecopies and, in one embodiment, is expressed in the host cell.

According to the invention, the term “expression” is used in its mostgeneral meaning and comprises the production of RNA or of RNA andprotein. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.Preferred expression systems in mammalian cells comprise pcDNA3.1 andpRc/CMV (Invitrogen, Carlsbad, Calif.), which contain a selectablemarker such as a gene imparting resistance to G418 (and thus enablingstably transfected cell lines to be selected) and the enhancer-promotersequences of cytomegalovirus (CMV).

In those cases of the invention in which a MHC molecule presents atumor-associated antigen or a part thereof, an expression vector mayalso comprise a nucleic acid sequence coding for said MHC molecule. Thenucleic acid sequence coding for the MHC molecule may be present on thesame expression vector as the nucleic acid coding for thetumor-associated antigen or the part thereof, or both nucleic acids maybe present on different expression vectors. In the latter case, the twoexpression vectors may be cotransfected into a cell. If a host cellexpresses neither the tumor-associated antigen or the part thereof northe MHC molecule, both nucleic acids coding therefor may be transfectedinto the cell either on the same expression vector or on differentexpression vectors. If the cell already expresses the MHC molecule, onlythe nucleic acid sequence coding for the tumor-associated antigen or thepart thereof can be transfected into the cell.

The invention also comprises kits for amplification of a nucleic acidcoding for a tumor-associated antigen. Such kits comprise, for example,a pair of amplification primers which hybridize to the nucleic acidcoding for the tumor-associated antigen. The primers preferably comprisea sequence of 6-50, in particular 10-30, 15-30 and 20-30 contiguousnucleotides of the nucleic acid and are nonoverlapping, in order toavoid the formation of primer dimers. One of the primers will hybridizeto one strand of the nucleic acid coding for the tumor-associatedantigen, and the other primer will hybridize to the complementary strandin an arrangement which allows amplification of the nucleic acid codingfor the tumor-associated antigen.

“Antisense molecules” or “antisense nucleic acids” may be used forregulating, in particular reducing, expression of a nucleic acid. Theterm “antisense molecule” or “antisense nucleic acid” refers accordingto the invention to an oligonucleotide which is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide or modifiedoligodeoxyribonucleotide and which hybridizes under physiologicalconditions to DNA comprising a particular gene or to mRNA of said gene,thereby inhibiting transcription of said gene and/or translation of saidmRNA. According to the invention, an “antisense molecule” also comprisesa construct which contains a nucleic acid or a part thereof in reverseorientation with respect to its natural promoter. An antisensetranscript of a nucleic acid or of a part thereof may form a duplex withthe naturally occurring mRNA specifying the enzyme and thus preventaccumulation of or translation of the mRNA into the active enzyme.Another possibility is the use of ribozymes for inactivating a nucleicacid. Antisense oligonucleotides preferred according to the inventionhave a sequence of 6-50, in particular 10-30, 15-30 and 20-30,contiguous nucleotides of the target nucleic acid and preferably arefully complementary to the target nucleic acid or to a part thereof.

In preferred embodiments, the antisense oligonucleotide hybridizes withan N-terminal or 5′ upstream site such as a translation initiation site,transcription initiation site or promoter site. In further embodiments,the antisense oligonucleotide hybridizes with a 3′ untranslated regionor mRNA splicing site.

In one embodiment, an oligonucleotide of the invention consists ofribonucleotides, deoxyribonucleotides or a combination thereof, with the5′ end of one nucleotide and the 3′ end of another nucleotide beinglinked to one another by a phosphodiester bond. These oligonucleotidesmay be synthesized in the conventional manner or produced recombinantly.

In preferred embodiments, an oligonucleotide of the invention is a“modified” oligonucleotide. Here, the oligonucleotide may be modified invery different ways, without impairing its ability to bind its target,in order to increase, for example, its stability or therapeuticefficacy. According to the invention, the term “modifiedoligonucleotide” means an oligonucleotide in which (i) at least two ofits nucleotides are linked to one another by a synthetic internucleosidebond (i.e. an internucleoside bond which is not a phosphodiester bond)and/or (ii) a chemical group which is usually not found in nucleic acidsis covalently linked to the oligonucleotide. Preferred syntheticinternucleoside bonds are phosphorothioates, alkyl phosphonates,phosphorodithioates, phosphate esters, alkyl phosphonothioates,phosphoramidates, carbamates, carbonates, phosphate triesters,acetamidates, carboxymethyl esters and peptides.

The term “modified oligonucleotide” also comprises oligonucleotideshaving a covalently modified base and/or sugar. “Modifiedoligonucleotides” comprise, for example, oligonucleotides with sugarresidues which are covalently bound to low molecular weight organicgroups other than a hydroxyl group at the 3′ position and a phosphategroup at the 5′ position. Modified oligonucleotides may comprise, forexample, a 2′-O-alkylated ribose residue or another sugar instead ofribose, such as arabinose.

It is to be understood that all embodiments described above with respectto oligonucleotides may also apply to polynucleotides.

By “small interfering RNA” or “siRNA” as used herein is meant anisolated RNA molecule, preferably greater than 10 nucleotides in length,more preferably greater than 15 nucleotides in length, and mostpreferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides in length that is used to identify a target gene or mRNA tobe degraded. A range of 19-25 nucleotides is the most preferred size forsiRNAs.

siRNA according to the invention can comprise partially purified RNA,substantially pure RNA, synthetic RNA, or recombinantly produced RNA, aswell as altered RNA that differs from naturally-occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the siRNA or to one or more internalnucleotides of the siRNA; modifications that make the siRNA resistant tonuclease digestion (e.g., the use of 2′-substituted ribonucleotides ormodifications to the sugar-phosphate backbone); or the substitution ofone or more nucleotides in the siRNA with deoxyribonucleotides.Furthermore, siRNA may be modified to increase the stability thereof asdescribed above for modified oligonucleotides, in particular byintroducing one or more phosphorothioate linkages.

One or both strands of the siRNA can also comprise a 3′-overhang. Asused herein, a “3′-overhang” refers to at least one unpaired nucleotideextending from the 3′-end of an RNA strand. Thus in one embodiment, thesiRNA comprises at least one 3′-overhang of from 1 to about 6nucleotides (which includes ribonucleotides or deoxynucleotides) inlength, preferably from 1 to about 5 nucleotides in length, morepreferably from 1 to about 4 nucleotides in length, and particularlypreferably from about 2 to about 4 nucleotides in length. In theembodiment in which both strands of the siRNA molecule comprise a3′-overhang, the length of the overhangs can be the same or differentfor each strand. In a most preferred embodiment, the 3′-overhang ispresent on both strands of the siRNA, and is 2 nucleotides in length.For example, each strand of the siRNA of the invention can comprise3′-overhangs of dideoxythymidylic acid (“TT”) or diuridylic acid (“uu”).

In order to enhance the stability of the siRNA, the 3′-overhangs can bealso stabilized against degradation. In one embodiment, the overhangsare stabilized by including purine nucleotides, such as adenosine orguanosine nucleotides. Alternatively, substitution of pyrimidinenucleotides by modified analogues, e.g., substitution of uridinenucleotides in the 3′-overhangs with 2′-deoxythymidine, is tolerated anddoes not affect the efficiency of RNAi degradation. In particular, theabsence of a 2′-hydroxyl in the 2′-deoxythymidine significantly enhancesthe nuclease resistance of the 3′-overhang in tissue culture medium.

The sense and antisense strands of the siRNA can comprise twocomplementary, single-stranded RNA molecules or can comprise a singlemolecule in which two complementary portions are base-paired and arecovalently linked by a single-stranded “hairpin” area. That is, thesense region and antisense region can be covalently connected via alinker molecule. The linker molecule can be a polynucleotide ornon-nucleotide linker. Without wishing to be bound by any theory, it isbelieved that the hairpin area of the latter type of siRNA molecule iscleaved intracellularly by the “Dicer” protein (or its equivalent) toform a siRNA of two individual base-paired RNA molecules.

As used herein, “target mRNA” refers to an RNA molecule that is a targetfor downregulation.

siRNA can be expressed from pol III expression vectors without a changein targeting site, as expression of RNAs from pol III promoters is onlybelieved to be efficient when the first transcribed nucleotide is apurine.

siRNA according to the invention can be targeted to any stretch ofapproximately 19-25 contiguous nucleotides in any of the target mRNAsequences (the “target sequence”). Techniques for selecting targetsequences for siRNA are given, for example, in Tuschl T. et al., “ThesiRNA User Guide”, revised Oct. 11, 2002, the entire disclosure of whichis herein incorporated by reference. “The siRNA User Guide” is availableon the world wide web at a website maintained by Dr. Thomas Tuschl,Laboratory of RNA Molecular Biology, Rockefeller University, New York,USA, and can be found by accessing the website of the RockefellerUniversity and searching with the keyword “siRNA”. Thus, the sensestrand of the present siRNA comprises a nucleotide sequencesubstantially identical to any contiguous stretch of about 19 to about25 nucleotides in the target mRNA.

Generally, a target sequence on the target mRNA can be selected from agiven cDNA sequence corresponding to the target mRNA, preferablybeginning 50 to 100 nt downstream (i.e., in the 3′-direction) from thestart codon. The target sequence can, however, be located in the 5′- or3′-untranslated regions, or in the region nearby the start codon.

siRNA can be obtained using a number of techniques known to those ofskill in the art. For example, siRNA can be chemically synthesized orrecombinantly produced using methods known in the art, such as theDrosophila in vitro system described in U.S. published application2002/0086356 of Tuschl et al., the entire disclosure of which is hereinincorporated by reference.

Preferably, siRNA is chemically synthesized using appropriatelyprotected ribonucleoside phosphoramidites and a conventional DNA/RNAsynthesizer. siRNA can be synthesized as two separate, complementary RNAmolecules, or as a single RNA molecule with two complementary regions.

Alternatively, siRNA can also be expressed from recombinant circular orlinear DNA plasmids using any suitable promoter. Such embodiments areincluded according to the present invention when reference is madeherein to the administration of siRNA or the incorporation of siRNA intopharmaceutical compositions. Suitable promoters for expressing siRNA ofthe invention from a plasmid include, for example, the U6 or H1 RNA polIII promoter sequences and the cytomegalovirus promoter.

Selection of other suitable promoters is within the skill in the art.The recombinant plasmids of the invention can also comprise inducible orregulatable promoters for expression of the siRNA in a particular tissueor in a particular intracellular environment.

The siRNA expressed from recombinant plasmids can either be isolatedfrom cultured cell expression systems by standard techniques, or can beexpressed intracellularly. The use of recombinant plasmids to deliversiRNA to cells in vivo is discussed in more detail below. siRNA can beexpressed from a recombinant plasmid either as two separate,complementary RNA molecules, or as a single RNA molecule with twocomplementary regions.

Selection of plasmids suitable for expressing siRNA, methods forinserting nucleic acid sequences for expressing the siRNA into theplasmid, and methods of delivering the recombinant plasmid to the cellsof interest are within the skill in the art.

siRNA can also be expressed from recombinant viral vectorsintracellularly in vivo. The recombinant viral vectors comprisesequences encoding the siRNA and any suitable promoter for expressingthe siRNA sequences. The recombinant viral vectors can also compriseinducible or regulatable promoters for expression of the siRNA in aparticular tissue or in a particular intracellular environment. siRNAcan be expressed from a recombinant viral vector either as two separate,complementary RNA molecules, or as a single RNA molecule with twocomplementary regions.

The term “peptide” comprises oligo- and polypeptides and refers tosubstances comprising two or more, preferably 3 or more, preferably 4 ormore, preferably 6 or more, preferably 8 or more, preferably 10 or more,preferably 13 or more, preferably 16 more, preferably 21 or more and upto preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acidsjoined covalently by peptide bonds. The term “protein” refers to largepeptides, preferably to peptides with more than 100 amino acid residues,but in general the terms “peptides” and “proteins” are synonyms and areused interchangeably herein.

Preferably, the proteins and peptides described according to theinvention have been isolated. The terms “isolated protein” or “isolatedpeptide” mean that the protein or peptide has been separated from itsnatural environment. An isolated protein or peptide may be in anessentially purified state. The term “essentially purified” means thatthe protein or peptide is essentially free of other substances withwhich it is associated in nature or in vivo.

Such proteins and peptides may be used, for example, in producingantibodies and in an immunological or diagnostic assay or astherapeutics. Proteins and peptides described according to the inventionmay be isolated from biological samples such as tissue or cellhomogenates and may also be expressed recombinantly in a multiplicity ofpro- or eukaryotic expression systems.

For the purposes of the present invention, “derivatives” of a protein orpeptide or of an amino acid sequence comprise amino acid insertionvariants, amino acid deletion variants and/or amino acid substitutionvariants.

Amino acid insertion variants comprise amino- and/or carboxy-terminalfusions and also insertions of single or two or more amino acids in aparticular amino acid sequence. In the case of amino acid sequencevariants having an insertion, one or more amino acid residues areinserted into a particular site in an amino acid sequence, althoughrandom insertion with appropriate screening of the resulting product isalso possible. Amino acid deletion variants are characterized by theremoval of one or more amino acids from the sequence. Amino acidsubstitution variants are characterized by at least one residue in thesequence being removed and another residue being inserted in its place.Preference is given to the modifications being in positions in the aminoacid sequence which are not conserved between homologous proteins orpeptides and/or to replacing amino acids with other ones having similarproperties such as hydrophobicity, hydrophilicity, electronegativity,volume of the side chain and the like (conservative substitution).Conservative substitutions, for example, relate to the exchange of oneamino acid with another amino acid listed below in the same group as theamino acid to be substituted:

-   1. small aliphatic, nonpolar or slightly polar residues: Ala, Ser,    Thr (Pro, Gly)-   2. negatively charged residues and their amides: Asn, Asp, Glu, Gln-   3. positively charged residues: His, Arg, Lys-   4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys)-   5. large aromatic residues: Phe, Tyr, Trp.

Owing to their particular part in protein architecture, three residuesare shown in brackets. Gly is the only residue without a side chain andthus imparts flexibility to the chain. Pro has an unusual geometry whichgreatly restricts the chain. Cys can form a disulfide bridge.

The amino acid variants described above may be readily prepared with theaid of known peptide synthesis techniques such as, for example, by solidphase synthesis (Merrifield, 1964) and similar methods or by recombinantDNA manipulation. The manipulation of DNA sequences for preparingproteins and peptides having substitutions, insertions or deletions, isdescribed in detail in Sambrook et al. (1989), for example.

According to the invention, “derivatives” of proteins and peptides alsocomprise single or multiple substitutions, deletions and/or additions ofany molecules associated with the protein or peptide, such ascarbohydrates, lipids and/or proteins or peptides. The term “derivative”also extends to all functional chemical equivalents of said proteins andpeptides.

According to the invention, a part or fragment of a tumor-associatedantigen preferably has a functional property of the protein or peptidefrom which it has been derived. Such functional properties comprise theinteraction with antibodies, the interaction with other peptides orproteins, the selective binding of nucleic acids and an enzymaticactivity. A particular property is the ability to form a complex withMHC molecules and, where appropriate, generate an immune response,preferably by stimulating cytotoxic or T helper cells. A part orfragment of a tumor-associated antigen of the invention preferablycomprises a sequence of at least 6, in particular at least 8, at least10, at least 12, at least 15, at least 20, at least 30 or at least 50,consecutive amino acids of the tumor-associated antigen. A part orfragment of a tumor-associated antigen of the invention preferablycomprises a sequence of up to 8, in particular up to 10, up to 12, up to15, up to 20, up to 30 or up to 55, consecutive amino acids of thetumor-associated antigen. A part or fragment of a tumor-associatedantigen is preferably a part of the tumor-associated antigen whichcorresponds to the non-transmembrane portion, in particular theextracellular portion of the antigen, or is comprised thereof.

Preferred parts or fragments of a tumor-associated antigen according tothe invention are in particular suitable for the stimulation ofcytotoxic T-lymphocytes in vivo but also for the production of expandedand stimulated T-lymphocytes for the therapeutic adoptive transfer exvivo.

A part or a fragment of a nucleic acid coding for a tumor-associatedantigen relates according to the invention to the part of the nucleicacid, which codes at least for the tumor-associated antigen and/or for apart or a fragment of said tumor-associated antigen, as defined above. Apart or fragment of a nucleic acid coding for a tumor-associated antigenis preferably that part of the nucleic acid corresponding to the openreading frame.

According to the invention, particular embodiments ought to involveproviding “dominant negative” proteins or peptides derived fromtumor-associated antigens. A dominant negative protein or peptide is aninactive protein or peptide variant which, by way of interacting withthe cellular machinery, displaces an active protein or peptide from itsinteraction with the cellular machinery or which competes with theactive protein or peptide, thereby reducing the effect of said activeprotein.

Antisera which contain specific antibodies specifically binding to thetarget protein can be prepared by various standard processes; see, forexample, “Monoclonal Antibodies: A Practical Approach” by PhilipShepherd, Christopher Dean ISBN 0-19-963722-9; “Antibodies: A LaboratoryManual” by Ed Harlow, David Lane, ISBN: 0879693142 and “UsingAntibodies: A Laboratory Manual: Portable Protocol NO” by Edward Harlow,David Lane, Ed Harlow ISBN 0879695447. Thereby it is also possible togenerate affine and specific antibodies which recognize complex membraneproteins in their native form (Azorsa et al., J. Immunol. Methods 229:35-48, 1999; Anderson et al., J. Immunol. 143: 1899-1904, 1989;Gardsvoll, J. Immunol. Methods 234: 107-116, 2000). This is inparticular relevant for the preparation of antibodies which are to beused therapeutically, but also for many diagnostic applications. In thisrespect, it is possible to immunize with the whole protein, withextracellular partial sequences as well as with cells which express thetarget molecule in physiologically folded form.

Monoclonal antibodies are traditionally prepared using the hybridomatechnology. (for technical details see: “Monoclonal Antibodies: APractical Approach” by Philip Shepherd, Christopher Dean ISBN0-19-963722-9; “Antibodies: A Laboratory Manual” by Ed Harlow, DavidLane ISBN: 0879693142; “Using Antibodies: A Laboratory Manual: PortableProtocol NO” by Edward Harlow, David Lane, Ed Harlow ISBN: 0879695447).

It is known that only a small part of an antibody molecule, theparatope, is involved in binding of the antibody to its epitope (cf.Clark, W. R. (1986), The Experimental Foundations of Modern Immunology,Wiley & Sons, Inc., New York; Roitt, I. (1991), Essential Immunology,7th Edition, Blackwell Scientific Publications, Oxford). The pFc′ and Fcregions are, for example, effectors of the complement cascade but arenot involved in antigen binding. An antibody from which the pFc′ regionhas been enzymatically removed or which has been produced without thepFc′ region, referred to as F(ab′)₂ fragment, carries both antigenbinding sites of a complete antibody. Similarly, an antibody from whichthe Fc region has been enzymatically removed or which has been producedwithout said Fc region, referred to as Fab fragment, carries one antigenbinding site of an intact antibody molecule. Furthermore, Fab fragmentsconsist of a covalently bound light chain of an antibody and part of theheavy chain of said antibody, referred to as Fd. The Fd fragments arethe main determinants of antibody specificity (a single Fd fragment canbe associated with up to ten different light chains, without alteringthe specificity of the antibody) and Fd fragments, when isolated, retainthe ability to bind to an epitope.

Located within the antigen-binding part of an antibody arecomplementary-determining regions (CDRs) which interact directly withthe antigen epitope and framework regions (FRs) which maintain thetertiary structure of the paratope. Both the Fd fragment of the heavychain and the light chain of IgG immunoglobulins contain four frameworkregions (FR1 to FR4) which are separated in each case by threecomplementary-determining regions (CDR1 to CDR3). The CDRs and, inparticular, the CDR3 regions and, still more particularly, the CDR3region of the heavy chain are responsible to a large extent for antibodyspecificity.

Non-CDR regions of a mammalian antibody are known to be able to bereplaced by similar regions of antibodies with the same or a differentspecificity, with the specificity for the epitope of the originalantibody being retained. This made possible the development of“humanized” antibodies in which nonhuman CDRs are covalently linked tohuman FR and/or Fc/pFc′ regions to produce a functional antibody.

As another example, WO 92/04381 describes the production and use ofhumanized murine RSV antibodies in which at least part of the murine FRregions have been replaced with FR regions of a human origin. Antibodiesof this kind, including fragments of intact antibodies withantigen-binding capability, are often referred to as “chimeric”antibodies.

According to the invention, the term “antibody” also includes F(ab′)₂,Fab, Fv, and Fd fragments of antibodies, chimeric antibodies, in whichthe Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain-CDR3 regionshave been replaced with homologous human or nonhuman sequences, chimericF(ab′)₂-fragment antibodies in which the FR and/or CDR1 and/or CDR2and/or light chain-CDR3 regions have been replaced with homologous humanor nonhuman sequences, chimeric Fab-fragment antibodies in which the FRand/or CDR1 and/or CDR2 and/or light chain-CDR3 regions have beenreplaced with homologous human or nonhuman sequences, and chimericFd-fragment antibodies in which the FR and/or CDR1 and/or CDR2 regionshave been replaced with homologous human or nonhuman sequences. The term“antibody” also comprises “single-chain” antibodies.

The invention also comprises proteins and peptides which bindspecifically to tumor-associated antigens. Binding substances of thiskind may be provided, for example, by degenerate peptide libraries whichmay be prepared simply in solution in an immobilized form or asphage-display libraries. It is likewise possible to preparecombinatorial libraries of peptides with one or more amino acids.Libraries of peptoids and nonpeptidic synthetic residues may also beprepared.

Antibodies may also be coupled to specific diagnostic substances fordisplaying cells and tissues expressing tumor-associated antigens. Theymay also be coupled to therapeutically useful substances.

Diagnostic substances include any label that functions to: (i) provide adetectable signal; (ii) interact with a second label to modify thedetectable signal provided by the first or second label, e.g. FRET(Fluorescence Resonance Energy Transfer); (iii) affect mobility, e.g.electrophoretic mobility, by charge, hydrophobicity, shape, or otherphysical parameters, or (iv) provide a capture moiety, e.g., affinity,antibody/antigen, or ionic complexation. Suitable as label arestructures, such as fluorescent labels, luminescent labels, chromophorelabels, radioisotopic labels, isotopic labels, preferably stableisotopic labels, isobaric labels, enzyme labels, particle labels, inparticular metal particle labels, magnetic particle labels, polymerparticle labels, small organic molecules such as biotin, ligands ofreceptors or binding molecules such as cell adhesion proteins orlectins, label-sequences comprising nucleic acids and/or amino acidresidues which can be detected by use of binding agents, etc. Diagnosticsubstances comprise, in a nonlimiting manner, barium sulfate, iocetamicacid, iopanoic acid, calcium ipodate, sodium diatrizoate, megluminediatrizoate, metrizamide, sodium tyropanoate and radio diagnostic,including positron emitters such as fluorine-18 and carbon-11, gammaemitters such as iodine-123, technetium-99m, iodine-131 and indium-111,nuclides for nuclear magnetic resonance, such as fluorine andgadolinium.

According to the invention, the term “therapeutically useful substance”means any molecule which may exert a therapeutic effect. According tothe invention, a therapeutically useful substance is preferablyselectively guided to a cell which expresses one or moretumor-associated antigens and includes anticancer agents, radioactiveiodine-labeled compounds, toxins, cytostatic or cytolytic drugs, etc.Anticancer agents comprise, for example, aminoglutethimide,azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil,cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine,dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil,interferon-α, lomustine, mercaptopurine, methotrexate, mitotane,procarbazine HCl, thioguanine, vinblastine sulfate and vincristinesulfate. Other anticancer agents are described, for example, in Goodmanand Gilman, “The Pharmacological Basis of Therapeutics”, 8th Edition,1990, McGraw-Hill, Inc., in particular Chapter 52 (Antineoplastic Agents(Paul Calabresi and Bruce A. Chabner). Toxins may be proteins such aspokeweed antiviral protein, cholera toxin, pertussis toxin, ricin,gelonin, abrin, diphtheria exotoxin or Pseudomonas exotoxin. Toxinresidues may also be high energy-emitting radionuclides such ascobalt-60.

The term “major histocompatibility complex” or “MHC” relates to acomplex of genes present in all vertebrates. MHC proteins or moleculesare involved in signaling between lymphocytes and antigen presentingcells in normal immune reactions by binding peptides and presenting themfor recognition by T cell receptors (TCR). MHC molecules bind peptideswithin an intracellular processing compartment and present thesepeptides on the surface of antigen presenting cells for recognition by Tcells. The human MHC region also termed HLA is located on chromosome 6and includes the class I and class II region. In one preferredembodiment of all aspects of the invention an MHC molecule is an HLAmolecule.

“Reduce” or “inhibit” as used herein means the ability to cause anoverall decrease, preferably of 20% or greater, more preferably of 50%or greater, and most preferably of 75% or greater, in the level, e.g. inthe level of protein or mRNA as compared to a reference sample (e.g., asample not treated with siRNA). This reduction or inhibition of RNA orprotein expression can occur through targeted mRNA cleavage ordegradation. Assays for protein expression or nucleic acid expressionare known in the art and include, for example, ELISA, western blotanalysis for protein expression, and northern blotting or RNaseprotection assays for RNA.

The term “patient” means according to the invention a human being, anonhuman primate or another animal, in particular a mammal such as acow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouse andrat. In a particularly preferred embodiment, the patient is a humanbeing.

“Abnormal expression” means according to the invention that expressionis altered, preferably increased, compared to the state in a healthyindividual.

According to the invention the term “increased” or “increased amount”preferably refers to an increase by at least 10%, in particular at least20%, at least 50% or at least 100%. The amount of a substance is alsoincreased in a test sample such as a biological sample compared to areference sample if it is detectable in the test sample but absent ornot detectable in the reference sample.

According to the invention, the term “disease” refers to anypathological state in which tumor-associated antigens are expressed orabnormally expressed. “Abnormal expression” means according to theinvention that expression is altered, preferably increased, compared tothe state in a healthy individual. An increase in expression refers toan increase by at least 10%, in particular at least 20%, at least 50% orat least 100%. In one embodiment, the tumor-associated antigen isexpressed only in tissue of a diseased individual, while expression in ahealthy individual is repressed. One example of such a disease iscancer, wherein the term “cancer” according to the invention comprisesleukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas,gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenalcancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain,cervical cancer, intestinal cancer, liver cancer, colon cancer, stomachcancer, intestine cancer, head and neck cancer, gastrointestinal cancer,lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer,ear, nose and throat (ENT) cancer, breast cancer, prostate cancer,cancer of the uterus, ovarian cancer and lung cancer and the matastasesthereof. Examples thereof are lung carcinomas, mamma carcinomas,prostate carcinomas, colon carcinomas, renal cell carcinomas, cervicalcarcinomas, or metastases of the cancer types or tumors described above.The term cancer according to the invention also comprises cancermetastases.

By “tumor” is meant an abnormal group of cells or tissue that grows by arapid, uncontrolled cellular proliferation and continues to grow afterthe stimuli that initiated the new growth cease. Tumors show partial orcomplete lack of structural organization and functional coordinationwith the normal tissue, and usually form a distinct mass of tissue,which may be either benign or malignant.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor at the target site depends onangiogenesis. Tumor metastasis often occurs even after the removal ofthe primary tumor because tumor cells or components may remain anddevelop metastatic potential. In one embodiment, the term “metastasis”according to the invention relates to “distant metastasis” which relatesto a metastasis which is remote from the primary tumor and the regionallymph node system.

According to the invention, a biological sample may be a tissue sample,including bodily fluids, and/or a cellular sample and may be obtained inthe conventional manner such as by tissue biopsy, including punchbiopsy, and by taking blood, bronchial aspirate, sputum, urine, feces orother body fluids. According to the invention, the term “biologicalsample” also includes fractions of biological samples.

According to the invention, the term “immunoreactive cell” means a cellwhich can mature into an immune cell (such as B cell, T helper cell, orcytolytic T cell) with suitable stimulation. Immunoreactive cellscomprise CD34⁺ hematopoietic stem cells, immature and mature T cells andimmature and mature B cells. If production of cytolytic or T helpercells recognizing a tumor-associated antigen is desired, theimmunoreactive cell is contacted with a cell expressing atumor-associated antigen under conditions which favor production,differentiation and/or selection of cytolytic T cells and of T helpercells. The differentiation of T cell precursors into a cytolytic T cell,when exposed to an antigen, is similar to clonal selection of the immunesystem.

The terms “T cell” and “T lymphocyte” are used interchangeably hereinand include T helper cells and cytotoxic T cells which comprisecytolytic T cells.

Some therapeutic methods are based on a reaction of the immune system ofa patient, which results in a lysis of antigen-presenting cells such ascancer cells which present one or more tumor-associated antigens. Inthis connection, for example autologous cytotoxic lymphocytes specificfor a complex of a tumor-associated antigen and an MHC molecule areadministered to a patient having a cellular abnormality. The productionof such cytotoxic T lymphocytes in vitro is known. An example of amethod of differentiating T cells can be found in WO-A-9633265.Generally, a sample containing cells such as blood cells is taken fromthe patient and the cells are contacted with a cell which presents thecomplex and which can cause propagation of cytotoxic T lymphocytes (e.g.dendritic cells). The target cell may be a transfected cell such as aCOS cell. These transfected cells present the desired complex on theirsurface and, when contacted with cytotoxic T lymphocytes, stimulatepropagation of the latter. The clonally expanded autologous cytotoxic Tlymphocytes are then administered to the patient.

In another method of selecting antigen-specific cytotoxic T lymphocytes,fluorogenic tetramers of MHC class I molecule/peptide complexes are usedfor obtaining specific clones of cytotoxic T lymphocytes (Altman et al.,Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416, 1998).

The present invention also includes therapeutic methods referred to asadoptive transfer (Greenberg, J. Immunol. 136(5):1917, 1986; Riddel etal., Science 257:238, 1992; Lynch et al., Eur. J. Immunol. 21:1403-1410,1991; Kast et al., Cell 59:603-614, 1989), wherein cells presenting thedesired complex (e.g. dendritic cells) are combined with cytotoxic Tlymphocytes of the patient to be treated, resulting in a propagation ofspecific cytotoxic T lymphocytes. The propagated cytotoxic T lymphocytesare then administered to a patient having a cellular anomalycharacterized by particular abnormal cells presenting the specificcomplex. The cytotoxic T lymphocytes then lyse the abnormal cells,thereby achieving a desired therapeutic effect.

Furthermore, cells presenting the desired complex (e.g. dendritic cells)may be combined with cytotoxic T lymphocytes of healthy individuals oranother species (e.g. mouse) which may result in propagation of specificcytotoxic T lymphocytes with high affinity. The high affinity T cellreceptor of these propagated specific T lymphocytes may be cloned andoptionally humanized to a different extent, and the T cell receptorsthus obtained then transduced via gene transfer, for example usingretroviral vectors, into T cells of patients. Adoptive transfer may thenbe carried out using these genetically altered T lymphocytes(Stanislawski et al., Nat. Immunol. 2:962-70, 2001; Kessels et al., Nat.Immunol. 2:957-61, 2001).

Adoptive transfer is not the only form of therapy which can be appliedaccording to the invention. Cytotoxic T lymphocytes may also begenerated in vivo in a manner known per se. One method usesnonproliferative cells expressing the complex. The cells used here willbe those which usually express the complex, such as irradiated tumorcells or cells transfected with one or both genes necessary forpresentation of the complex (i.e. the antigenic peptide and thepresenting MHC molecule). Another preferred form is the introduction ofthe tumor-associated antigen in the form of recombinant RNA which may beintroduced into cells by liposomal transfer or by electroporation, forexample. The resulting cells present the complex of interest and arerecognized by autologous cytotoxic T lymphocytes which then propagate.

A similar effect can be achieved by combining the tumor-associatedantigen or a fragment thereof with an adjuvant in order to makeincorporation into antigen-presenting cells in vivo possible. Thetumor-associated antigen or a fragment thereof may be represented asprotein, as DNA (e.g. within a vector) or as RNA. The tumor-associatedantigen is processed to produce a peptide partner for the MHC molecule,while a fragment thereof may be presented without the need for furtherprocessing. The latter is the case in particular, if these can bind toMHC molecules. Preference is given to administration forms in which thecomplete antigen is processed in vivo by a dendritic cell, since thismay also produce T helper cell responses which are needed for aneffective immune response (Ossendorp et al., Immunol Lett. 74:75-9,2000; Ossendorp et al., J. Exp. Med. 187:693-702, 1998). In general, itis possible to administer an effective amount of the tumor-associatedantigen to a patient by intradermal injection, for example. However,injection may also be carried out intranodally into a lymph node (Maloyet al., Proc Natl Acad Sci USA 98:3299-303, 2001).

The pharmaceutical compositions and methods of treatment describedaccording to the invention may also be used for immunization orvaccination to therapeutically treat or prevent a disease describedherein. According to the invention, the terms “immunization” or“vaccination” preferably relate to an increase in or activation of animmune response to an antigen. It is possible to use animal models fortesting an immunizing effect on cancer by using a tumor-associatedantigen or a nucleic acid coding therefor. For example, human cancercells may be introduced into a mouse to generate a tumor, and one ormore nucleic acids coding for tumor-associated antigens may beadministered. The effect on the cancer cells (for example reduction intumor size) may be measured as a measure for the effectiveness of animmunization by the nucleic acid.

As part of the composition for an immunization or a vaccination,preferably one or more tumor-associated antigens or stimulatingfragments thereof are administered together with one or more adjuvantsfor inducing an immune response or for increasing an immune response. Anadjuvant is a substance which is incorporated into the antigen oradministered together with the latter and which enhances the immuneresponse. Adjuvants may enhance the immune response by providing anantigen reservoir (extracellularly or in macrophages), activatingmacrophages and/or stimulating particular lymphocytes. Adjuvants areknown and comprise in a nonlimiting way monophosphoryl lipid A (MPL,SmithKline Beecham), saponins such as QS21 (SmithKline Beecham), DQS21(SmithKline Beecham; WO 96/33739), QS7, QS17, QS18 and QS-L1 (So et al.,Mol. Cells. 7:178-186, 1997), incomplete Freund's adjuvant, completeFreund's adjuvant, vitamin E, montanide, alum, CpG oligonucleotides (cf.Kreig et al., Nature 374:546-9, 1995) and various water-in-oil emulsionsprepared from biologically degradable oils such as squalene and/ortocopherol. Preferably, the peptides are administered in a mixture withDQS21/MPL. The ratio of DQS21 to MPL is typically about 1:10 to 10:1,preferably about 1:5 to 5:1 and in particular about 1:1. Foradministration to humans, a vaccine formulation typically contains DQS21and MPL in a range from about 1 μg to about 100 μg.

Other substances which stimulate an immune response of the patient mayalso be administered. It is possible, for example, to use cytokines in avaccination, owing to their regulatory properties on lymphocytes. Suchcytokines comprise, for example, interleukin-12 (IL-12) which was shownto increase the protective actions of vaccines (cf. Science268:1432-1434, 1995), GM-CSF and IL-18.

There are a number of compounds which enhance an immune response andwhich therefore may be used in a vaccination. Said compounds comprisecostimulating molecules provided in the form of proteins or nucleicacids such as B7-1 and B7-2 (CD80 and CD86, respectively).

The invention also provides for administration of nucleic acids,proteins or peptides. Proteins and peptides may be administered in amanner known per se. In one embodiment, nucleic acids are administeredby ex vivo methods, i.e. by removing cells from a patient, geneticmodification of said cells in order to incorporate a tumor-associatedantigen and reintroduction of the altered cells into the patient. Thisgenerally comprises introducing a functional copy of a gene into thecells of a patient in vitro and reintroducing the genetically alteredcells into the patient. The functional copy of the gene is under thefunctional control of regulatory elements which allow the gene to beexpressed in the genetically altered cells. Transfection andtransduction methods are known to the skilled worker. The invention alsoprovides for administering nucleic acids in vivo by using vectors suchas viruses and target-controlled liposomes. If according to theinvention reference is made to the administration or incorporation intopharmaceutical compositions of nucleic acids this includes embodimentswherein the nucleic acid is present in such vectors.

In a preferred embodiment, a virus or viral vector for administering anucleic acid coding for a tumor-associated antigen is selected from thegroup consisting of adenoviruses, adeno-associated viruses, pox viruses,including vaccinia virus and attenuated pox viruses, Semliki Forestvirus, retroviruses, Sindbis virus and Ty virus-like particles.Particular preference is given to adenoviruses and retroviruses. Theretroviruses are typically replication-deficient (i.e. they areincapable of generating infectious particles).

Methods of introducing nucleic acids into cells in vitro or in vivocomprise transfection of nucleic acid calcium phosphate precipitates,transfection of nucleic acids associated with DEAE, transfection orinfection with the above viruses carrying the nucleic acids of interest,liposome-mediated transfection, and the like. In particular embodiments,preference is given to directing the nucleic acid to particular cells.In such embodiments, a carrier used for administering a nucleic acid toa cell (e.g. a retrovirus or a liposome) may have a bound target controlmolecule. For example, a molecule such as an antibody specific for asurface membrane protein on the target cell or a ligand for a receptoron the target cell may be incorporated into or attached to the nucleicacid carrier. Preferred antibodies comprise antibodies which bindselectively a tumor-associated antigen. If administration of a nucleicacid via liposomes is desired, proteins binding to a surface membraneprotein associated with endocytosis may be incorporated into theliposome formulation in order to make target control and/or uptakepossible. Such proteins comprise capsid proteins or fragments thereofwhich are specific for a particular cell type, antibodies to proteinswhich are internalized, proteins addressing an intracellular site, andthe like.

The therapeutic compositions of the invention may be administered inpharmaceutically compatible preparations. Such preparations may usuallycontain pharmaceutically compatible concentrations of salts, buffersubstances, preservatives, carriers, supplementing immunity-enhancingsubstances such as adjuvants, e.g. CpG oligonucleotides, cytokines,chemokines, saponin, GM-CSF and/or RNA and, where appropriate, othertherapeutically active compounds.

The therapeutically active compounds of the invention may beadministered via any conventional route, including by injection orinfusion. The administration may be carried out, for example, orally,intravenously, intraperitonealy, intramuscularly, subcutaneously ortransdermally. Preferably, antibodies are therapeutically administeredby way of a lung aerosol. Antisense nucleic acids are preferablyadministered by slow intravenous administration.

The compositions of the invention are administered in effective amounts.An “effective amount” refers to the amount which achieves a desiredreaction or a desired effect alone or together with further doses. Inthe case of treatment of a particular disease or of a particularcondition characterized by expression of one or more tumor-associatedantigens, the desired reaction preferably relates to inhibition of thecourse of the disease. This comprises slowing down the progress of thedisease and, in particular, interrupting or reversing the progress ofthe disease. The desired reaction in a treatment of a disease or of acondition may also be delay of the onset or a prevention of the onset ofsaid disease or said condition. According to the invention, a diagnosisor treatment of cancer may also include the diagnosis or treatment ofcancer metastases which have already formed or will form. According tothe invention, the term “treatment” comprises therapeutic andprophylactic treatment, i.e. prevention.

An effective amount of a composition of the invention will depend on thecondition to be treated, the severeness of the disease, the individualparameters of the patient, including age, physiological condition, sizeand weight, the duration of treatment, the type of an accompanyingtherapy (if present), the specific route of administration and similarfactors.

The pharmaceutical compositions of the invention are preferably sterileand contain an effective amount of the therapeutically active substanceto generate the desired reaction or the desired effect.

The doses administered of the compositions of the invention may dependon various parameters such as the type of administration, the conditionof the patient, the desired period of administration, etc. In the casethat a reaction in a patient is insufficient with an initial dose,higher doses (or effectively higher doses achieved by a different, morelocalized route of administration) may be used.

Generally, doses of the tumor-associated antigen of from 1 ng to 1 mg,preferably from 10 ng to 100 μg, are formulated and administered for atreatment or for generating or increasing an immune response. If theadministration of nucleic acids (DNA and RNA) coding fortumor-associated antigens is desired, doses of from 1 ng to 0.1 mg areformulated and administered.

The pharmaceutical compositions of the invention are generallyadministered in pharmaceutically compatible amounts and inpharmaceutically compatible compositions. The term “pharmaceuticallycompatible” refers to a nontoxic material which does not interact withthe action of the active component of the pharmaceutical composition.Preparations of this kind may usually contain salts, buffer substances,preservatives, carriers and, where appropriate, other therapeuticallyactive compounds. When used in medicine, the salts should bepharmaceutically compatible. However, salts which are notpharmaceutically compatible may used for preparing pharmaceuticallycompatible salts and are included in the invention. Pharmacologicallyand pharmaceutically compatible salts of this kind comprise in anonlimiting way those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallycompatible salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

A pharmaceutical composition of the invention may comprise apharmaceutically compatible carrier. According to the invention, theterm “pharmaceutically compatible carrier” refers to one or morecompatible solid or liquid fillers, diluents or encapsulatingsubstances, which are suitable for administration to humans. The term“carrier” refers to an organic or inorganic component, of a natural orsynthetic nature, in which the active component is combined in order tofacilitate application. The components of the pharmaceutical compositionof the invention are usually such that no interaction occurs whichsubstantially impairs the desired pharmaceutical efficacy.

The pharmaceutical compositions of the invention may contain suitablebuffer substances such as acetic acid in a salt, citric acid in a salt,boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may, where appropriate, also containsuitable preservatives such as benzalkonium chloride, chlorobutanol,paraben and thimerosal.

The pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. Pharmaceuticalcompositions of the invention may be in the form of capsules, tablets,lozenges, solutions, suspensions, syrups, elixirs or in the form of anemulsion, for example.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The present invention is described in detail by the figures and examplesbelow, which are used only for illustration purposes and are not meantto be limiting. Owing to the description and the examples, furtherembodiments which are likewise included in the invention are accessibleto the skilled worker.

FIGURES

FIG. 1. ISC-468 mRNA expression

-   -   A. RT-PCR investigations with ISC-468-specfic primers showed no        significant expression within all tested normal tissues except        placenta.    -   B. ISC-468 mRNA expression in head and neck, liver, kidney and        colon carcinomas.    -   C. ISC-468 mRNA expression in breast, ovarian and stomach        carcinomas.

FIG. 2. Quantitative PCR analysis of ISC-468 mRNA expression in normalcontrol tissues and breast cancers

Real-time PCR investigation with ISC-468-specific primers showedselective mRNA expression in normal testis, placenta, stomach and PBMC,and in all breast carcinoma biopsies.

FIG. 3. Specific ISC-507 expression in normal testis and prostatecarcinoma

RT-PCR analysis with gene-specific ISC-507 primers shows cDNAamplification exclusively in normal testis (A) and in prostate carcinomabiopsies (B).

FIG. 4. Quantitative expression of ISC-507

Quantitative RT-PCR with ISC-507-specific primers showed selectiveexpression in testis, lymph node and prostate samples and prostatecancer samples.

FIG. 5. ISC-466 expression in normal testis and various tumor samples

RT-PCR analysis with ISC-466-specific primers showed no expressionwithin normal tissues except placenta (A), but expression in head andneck carcinoma biopsies and in kidney carcinoma biopsies (B). Distinctexpression was also detected in breast and lung carcinoma cell lines, aswell as in ovarian carcinoma cell lines (C and D).

FIG. 6. ISC-518 mRNA expression

RT-PCR analysis with ISC-518-specific primers showed no expressionwithin normal tissues except testis.

FIG. 7. Quantitative expression of ISC-518

Quantitative RT-PCR showed high and selective expression in normaltestis and in one liver carcinoma-pool.

FIG. 8. ISC-477 expression in normal and tumor tissues

RT-PCR investigations with ISC-477-specific primers showed selectiveexpression in placenta and ovary normal tissue (A) and high expressionin investigated stomach carcinomas (B), breast, colon and lungcarcinomas (C), as well as in ovarian and pancreas carcinoma samples(D).

FIG. 9. ISC-489 mRNA expression

RT-PCR investigations with ISC-489-specific primers showed selectiveexpression in placenta control tissue and additionally various levels ofexpression in lung carcinoma samples (A, C), stomach carcinomas (B, C),head and neck tumors (C) and liver carcinoma samples (C).

FIG. 10. ISC-461 expression in normal testis and various tumor samples

RT-PCR investigations with ISC-461-specific primers showed selectiveexpression in placenta control tissue and additionally various levels ofexpression in breast carcinomas and melanomas (B), as well as in breastcarcinoma, lung carcinoma and melanoma cell lines (C) and ovariancarcinoma cell lines (D).

FIG. 11. ISC-465 mRNA expression in placenta and cancer derived samples

RT-PCR investigations with ISC-465-specific primers showed selectiveexpression in placenta (A) and in some cell lines derived from breastcancer, melanoma, lung cancer or stomach cancer (B).

FIG. 12. Quantitative expression of Mem-030

-   A. Quantitative RT-PCR with Mem-030-specific primers showed a    significant overexpression in all investigated head and neck    carcinoma samples. The following normal tissues were analyzed:    bladder, brain, bone marrow, cervix, colon, duodenum, heart, lung,    lymph node, breast, muscle, ovary, PBMC, PBMC-activated, placenta,    prostate, retina, spleen, stomach, testis, thymus and tonsil.-   B. Prevalence of Mem-030 in esophageal, liver, uterus carcimomas and    melanoma derived tissues.

FIG. 13. Quantitative expression of Mem-055

Quantitative RT-PCR with Mem-055-specific primers show high andselective expression in normal control tissues and a significantoverexpression in stomach and lung cancer derived tissues (A). Mem-055is also overexpressed in liver carcinomas, ovarian carcinomas and breastcancer samples (B).

FIG. 14. Mem-062 mRNA expression

RT-PCR analysis with Mem-064-specific primers showed selectiveexpression in testis and weak expression in lung cancer derived tissues(A). Strong, significant expression levels of Mem-064 transcripts weredetectable in various ovarian tumors (B).

FIG. 15. Specific Mem-068 expression in normal testis and renal cellcarcinomas

RT-PCR analysis with gene-specific Mem-068 primers shows cDNAamplification in normal testis, weak in placenta (A), in renal cellcarcinomas and in stomach cancers (B).

FIG. 16. Mem-071 expression in normal testis and various tumor samples

RT-PCR analysis with Mem-071-specific primers showed no expressionwithin normal tissues except testis (A). Distinct expression was alsodetected in renal cell carcinoma samples and in stomach cancers (B).

FIG. 17. Mem-072 mRNA expression

RT-PCR analysis with Mem-072-specific primers showed no expressionwithin normal tissues (A) and significant expression in various lungcancer samples (A+B).

FIG. 18. Mem-106 expression in normal and tumor tissues

RT-PCR investigations with Mem-106-specific primers showed no expressionwithin normal tissues except in testis (A) and high expression wereinvestigated in ovarian- and prostate carcinomas, as well as inmelanomas and colon cancer cellines (B).

FIG. 19. Mem-131 mRNA expression

RT-PCR investigations with Mem-131-specfic primers showed no significantexpression within all tested normal tissues except activatedPBMC.Mem-131 mRNA expression in breast- and lung-carcinomas.Mem-131 mRNAexpression in lung- and ovarian carcinomas.

FIG. 20. ISC-468 mRNA expression

(A) RT-PCR and B) Real-time PCR (investigation with ISC-468-specificprimers showed selective mRNA expression in normal testis, placenta, andin 80% of breast carcinoma biopsies.

FIG. 21. Immunofluorescence analysis of ISC-468 expression

(A) Specificity of anti-ISC-468 antibodies were confirmed by staining ofISC-468-eGFP transfected cells. (B) Staining of MeOH-fixed cells eithertransfected with ISC-468-specific RNAi duplexes, or non-silencingcontrol duplexes. (C) Staining of non-fixed cells either transfectedwith ISC-468-specific RNAi duplexes, or non-silencing control duplexes.

FIG. 22. Immunochistochmical analysis of ISC-468 expression

No expression was detectable in normal breast tissue (A) 100×, (B) 200×.In contrast, strong and homogeneous membran-staining was observed inbreast carcinoma specimens (C) 100×, (D) 200×.

FIG. 23. RNAi-induced knock-down of ISC-468 mRNA expression

Transfection of cells with ISC-468-specific siRNA duplexes resulted indistinct knock-down of ISC-468 mRNA expression compared to controlcells.

FIG. 24. Cell proliferation analysis

Transfection of cells with ISC-468-specific siRNA duplexes resulted indistinct impairement of cell proliferation compared to control cells.

FIG. 25. Cell cycle analysis

Transfection of cells with ISC-468-specific siRNA duplexes resulted inG1/S arrest in (A) MCF-7 and (B) BT-549 breast carcinoma cells comparedto control cells.

FIG. 26. AKT phosphorylierung

Transfection of cells with ISC-468-specific siRNA duplexes resulted indistinct impairement of AKT phosphorylation compared to control cells.

FIG. 27. Antibody-mediated proliferation inhibition

Incubation of MCF-7 breast carcinoma cells with ISC-468 specificantibodies resulted in reduced proliferation compared to cells incubatedwith an irrelevant control antibody.

FIG. 28. Cell proliferation analysis

Transfection of cells with ISC-468-specific siRNA duplexes resulted indistinct impairement of (A) chemotaxis, (B) chemokinesis, and (C)invasion compared to control cells.

FIG. 29. Estrogen receptor correlation

Expression levels of ISC-468 mRNA in breast carcinoma samples correlateswith the estrogen receptor state. Shown are the median, 10^(th), and90^(th) percentiles with error bars.

FIG. 30. 17β-estradiol treatment

ISC-468 mRNA expression was induced by treatment of estrogen receptorpositive breast carcinoma cell line MCF-7 with 100 nM 1713-estradiol. Noinduction was seen in estrogen receptor negative cell line MDA-MB-231.

FIG. 31. Sequences

The sequences to which reference is made herein are shown.

EXAMPLES Material and Methods

The techniques and methods mentioned herein are carried out in a mannerknown per se and are described, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. All methods includingthe use of kits and reagents are carried out according to themanufacturers' information.

RNA Extraction, Preparation of Poly-d(T) Primed cDNA and ConventionalRT-PCR Analysis

Total RNA was extracted from native tissue material by using guanidiumisothiocyanate as chaotropic agent (Chomczynski & Sacchi, Anal. Biochem.162:156-9, 1987). After extraction with acidic phenol and precipitationwith isopropanol, said RNA was dissolved in DEPC-treated water.

First strand cDNA synthesis from 4 μg of total RNA was carried out in a20 μl reaction mixture by means of Superscript II (Invitrogen),according to the manufacturer's information. The primer used was adT(18) oligonucleotide. Integrity and quality of the cDNA were checkedby amplification of p53 in a 30 cycle PCR ((SEQ ID NO:33,34)),hybridization temperature 67° C.)

An archive of first strand cDNA was prepared from a number of normaltissues and tumor entities. For expression studies, 0.5 μl of thesecDNAs was amplified in a 30 μl reaction mixture, using GOI-specificprimers (see below) and 1 U of HotStarTaq DNA polymerase (Qiagen). Eachreaction mixture contained 150 μM dNTPs, 0.3 μM of each primer and 3 μlof 10× reaction buffer. The primers were selected so as to be located intwo different exons, and elimination of the interference bycontaminating genomic DNA as the reason for false-positive results wasconfirmed by testing nonreverse-transcribed DNA as template. After 15minutes at 95° C. to activate the HotStarTaq DNA polymerase, 35 cyclesof PCR were carried out (0.5 min at 94° C., 0.5 min at the particularhybridization temperature, 0.5 min at 72° C. and final elongation at 72°C. for 6 min).

20 μl of this reaction were fractionated and analyzed on an ethidiumbromide-stained agarose gel.

Preparation of Random Hexamer-Primed cDNA and Quantitative Real-Time PCR

The expression of several genes was quantified by real-time PCR. The PCRproducts were detected using SYBR Green as intercalating reporter dye.The reporter fluorescence of SYBR Green is suppressed in solution andthe dye is active only after binding to double-stranded DNA fragments.The increase in the SYBR Green fluorescence as a result of the specificamplification using GOI-specific primers after each PCR cycle isutilized for quantification. Expression of the target gene is quantifiedabsolutely or relative to the expression of a control gene with constantexpression in the tissues to be investigated. Expression was measuredafter standardization of the samples against 18s RNA as so-calledhousekeeping gene using the ΔΔ-C_(t) method (PE Biosystems, USA). Thereactions were carried out in duplicates and determined in triplicates.The QuantiTect SYBR Green PCR kit (Qiagen, Hilden) was used inaccordance with the manufacturer's instructions. The cDNA wassynthesized with random primers (Invitrogen) using the protocoldescribed above. Each 5 μl portions of the diluted cDNA were employed ina total volume of 30 μl for the PCR: sense primer 300 nM, antisenseprimer 300 nM; initial denaturation 95° C. for 15 min; 95° C. for 30sec; annealing for 30 sec; 72° C. for 30 sec; 40 cycles. The sequencesof the primers used are indicated in the respective examples.

Cloning and Sequence Analysis

Cloning of full-lengths and gene fragments took place by conventionalmethods. To ascertain the sequence, corresponding antigenes wereamplified using the proofreading polymerase pfu (Stratagene). Aftercompletion of the PCR, adenosine was ligated by means of HotStarTaq DNApolymerase to the ends of the amplicon in order to clone the fragmentsin accordance with the manufacturer's instructions into the TOPO-TAvector. The sequencing was carried out by a commercial service. Thesequences were analysed using conventional prediction programs andalgorithms.

Cell Proliferation Analysis

24 h after transfection with siRNA duplexes 1×10⁴ cells were cultured inmedium supplemented with varying concentrations of FCS for 48 h.Proliferation was analyzed by measuring the incorporation of BrdU intonewly synthesized DNA strands using the DELFIA cell proliferation Kit(Perkin Elmer) according to the manufacturer's instructions on a WallacVictor2 multi-label counter (Perkin Elmer).

Cell Cycle Analysis and Apoptosis

Cells were cultured in medium supplemented with FCS in varyingconcentrations, harvested after 48 h and stained with propidiumiodideprior to flowcytometric DNA content analysis. Apoptotic cells and cellsin S/G2/M phases of the cell cycle were quantified usingCellQuest-Software (Becton Dickinson).

Cell Migration

Cell migration assays were conducted in transwell chambers with 8.0 μmpore membranes (BD Biosciences) with cells cultured in serum-free mediumfor 12 h prior to the experiments. For siRNA experiments cells weretransferred to serum-free conditions 24 h after transfection with siRNAduplexes as described above. 4×10⁴ cells in 400 μl serum-free culturemedium were added to the upper chamber. The bottom chambers contained800 μl culture medium supplemented with either FCS, PDGF-BB(Sigma-Aldrich) or SDF-1α/CXCL12 (R&D Systems) as chemoattractants.After 24 hours cells that had migrated to the bottom side of themembrane were fixed in ice-cold methanol; membranes were excised, placedon microscope slides and mounted with Hoechst (Dako) for fluorescencemicroscopy. Cells in five random visual fields (100× magnification) werecounted for each membrane. All experiments were done in triplicates.Effects on chemokinesis of cells was analyzed using the sameexperimental setup with (i) no chemoattractant added to the upper andlower chamber and (ii) with chemoattractant added to both the upper andlower chamber.

In Vitro Invasion Assay

In vivo invasion assays were conducted in transwell chambers with 8.0 μmpore membranes (BD Biosciences) with cells cultured in serum-free mediumfor 12 h prior to the experiments. Upper chambers were prepared with 100μl of Matrigel (BD Biosciences) diluted to 1 mg/ml in serum free medium.Chambers were incubated at 37° C. for 5 h for gelling. For siRNAexperiments cells were transferred to serum-free conditions 24 h aftertransfection with siRNA duplexes as described above. 1×10⁵ cells in 400μl serum-free culture medium were added to the upper chamber. The bottomchambers contained 800 μl culture medium supplemented with FCS aschemoattractant. After 24 hours invaded cells at the bottom side of themembrane were fixed in ice-cold methanol; membranes were excised, placedon microscope slides and mounted with Hoechst (Dako) for fluorescencemicroscopy. Cells in five random visual fields (100× magnification) werecounted for each membrane. All experiments were done in triplicates.

Example 1 Identification of ISC-468 as Therapeutic and Diagnostic CancerTarget

ISC-468 (SEQ ID NO:1) encodes a protein of 212 amino acids (SEQ ID NO:2)and with a molecular weight of 23.6 kDa.

It has been previously described as placenta-specific protein expressedduring pregnancy (Fant et al., Mol Reprod Dev. 63:430-6, 2002)

The protein is predicted to have a cleavable signal peptide from aa1-23, followed by a short putative transmembrane domain (aa 25-47) asanalysed by bioinformatics tools (TMpred, SOUSI). The remaining proteinis predicted to be extracellular and can therefore be used according tothe invention as target structure for monoclonal antibodies.

According to the invention, a gene-specific primer pair (SEQ ID NO:3, 4)for ISC-468 was used in RT-PCR analyses to amplify cDNA derived from acomprehensive panel of normal and tumor tissues. As expected, placentawas confirmed as the only healthy tissue expressing this gene (FIG. 1).No significant expression, whatsoever, was detected in any other normalorgan tissue. Most surprisingly, when cancer specimen were investigated,we found high and significant levels of expression in a number ofdifferent tumor types, including colon, pancreatic, esophageal, stomach,lung, breast, ovrian, head & neck, kidney, prostate and liver carcinomas(FIGS. 1 and 2 as well as tab.1). Quantitative real-time RT-PCR analysisof ISC-468 expression in 60 breast carcinoma samples revealed that 80%of all samples expressed significant levels of ISC-468 (FIG. 20A,B).

TABLE 1 ISC-468 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma + Myocardium −Pancreatic + carcinoma Skeletal muscle − Esophageal + carcinomaMyocardium − Stomach carcinoma + Stomach − Lung cancer + Colon − Breastcancer +++ Pancreas − Ovarian carcinoma + Kidney − Head & Neck Cancer +Liver − Kidney cancer + Testis − Prostate carcinoma + Thymus − Livercarcinoma ++ Breast − Ovary − Uterus − Skin − Lung − Placenta +++ Lymphnodes − Spleen − PBMC − Prostate −

The selective and high expression of ISC-468 transcripts in tumors wasnot previously known and can be utilized according to the invention formolecular diagnostic methods such as RT-PCR for detecting disseminatingtumor cells in the serum and bone marrow and for detecting metastases inother tissues. This molecule can be further used as specific target fortherapeutic approaches.

The following peptides, inter alia, were selected for producing ISC-468specific antibodies according to the invention: SEQ ID NO:58, 59, 60,68, 69, 2. Specificity of the antibodies was confirmed byimmunofluorescence analysis of ISC-468-eGFP transfected cells (FIG.21A).

The subcellular localization of ISC-468 in endogenously expressingbreast carcinoma cell lines MCF-7 and BT-549 was analyzed byimmunofluorescence analyses. Staining of either MeOH-fixed (FIG. 21B) ornon-fixed (FIG. 21C) cells revealed that ISC-468 is localized at theplasma membranes of the expressing cells. Specificity of the stainingwas confirmed by RNAi-induced knock-down of ISC-468 expression,resulting in the loss of plasma membrane staining.

Furthermore, ISC-468 specific antibodies were used forimmunohistochemical analysis of ISC-468 expression in clinical samplesof normal breast and breast carcinomas. Expression of ISC-468 was notdetectable in normal breast specimens (FIG. 22A,B). In contrast, breastcarcinoma specimens showed strong and homogeneous expression of ISC-468(FIG. 22C,D). Signals were accentuated at the plasmamembrane of theexpressing cancer cells, confirming that ISC-468 is a membrane proteinselectively expressed in cancer cells.

The extracellular domains of ISC-468 can be used according to theinvention as target structure for immunodiagnosis and therapy by meansof monoclonal antibodies. In addition, ISC-468 can be employed accordingto the invention as vaccine (RNA, DNA, protein, peptides) for inducingtumor-specific immune responses (T and B cell-mediated immuneresponses).

RNAi-induced knock-down of ISC-468 expression was achieved bytransfection of cells with siRNA duplexes specifically targeting ISC-468mRNA (SEQ ID NOs: 70-73). Transfection of endogenously expressing breastcarcinoma cell lines MCF-7 and BT-549 resulted in stable and specificreduction of ISC-468 mRNA expression (FIG. 23).

To gain insight into the physiological role of ISC-468 expressionseveral RNAi-based in vitro cell assays were performed. Transfection ofbreast carcionoma cell lines MCF-7 and BT-549 with siRNA duplexesresulted in a distinct reduction of cell proliferation compared to therespective controls, as analyzed in a BrdU-based proliferation assay(FIG. 24). FACS-based cell cycle analysis showed that the abrogation ofcell proliferation resulted from a G1/S arrest (FIG. 25A,B).Additionally, it could be shown that RNAi-induced knock-down of ISC-468profoundly affects the AKT signaling pathway in endogenously expressingcancer cells by inhibition of AKT phosphorylation (FIG. 26).Furthermore, proliferation of MCF-7 cells was attenuated when cells wereincubated with ISC-468 specific antibodies generated against ISC-468specific peptides (SEQ ID NO:68,69) compared to an irrelevant controlantibody (FIG. 27). These results indicate that ISC-468 is a criticalfactor for the proliferation of cancer cells presumably by mediatinggrowth factor-induced activation of the AKT signaling pathway andothers. ISC-468 itself might represent a receptor, co-receptor ormembrane-bound chaperone for growth-factors, chemokines or othersubstances.

Furthermore, the impact of ISC-468 expression on the migratory abilityof cancer cells was analyzed. RNAi-induced knock-down of ISC-468expression in breastcarcinoma cell lines MCF-7 and BT-549 resulted indistinct impairment of chemotaxis, chemokinesis and invasion of thecells, as assessed in transwell migration assays (FIG. 28A,B,C).Chemotaxis, chemokinesis and invasion are critical factors for themetastasis of cancer cells to other organs. Therefore, expression ofISC-468 in cancer cells might be a positive factor for cancer cellmetastasis.

In breast carcinomas, it could be shown that expression of ISC-468 iscorrelated with the estrogen-receptor state of the tumor. Quantitativereal-time RT-PCR analysis of ISC-468 expression in 60 breast carcinomasamples revealed that estrogen-receptor positive breast carcinomasshowed significantly higher levels of ISC-468 expression thanreceptor-negative tumors (FIG. 29).

Accordingly, expression of ISC-468 could be induced in estrogen-receptorpositive breast carcinoma cell line MCF-7 by treatment with178-estradiol (FIG. 30).

Example 2 Identification of ISC-507 as Therapeutic and Diagnostic CancerTarget

ISC-507 (SEQ ID NO:5) encodes a 754 aa protein (SEQ ID NO:6) with amolecular weight of 85.6 kDa. ISC-507 is member of a family ofzinc-binding proteins with disintegrin and metalloprotease activitiesthat can function as adhesion proteins and/or endopeptidases. Members ofthis family have been described as involved in a number of biologicprocesses, including fertilization, neurogenesis, muscle development,and immune response (Seals et al., Genes Dev. 17(1):7-30, 2003)

ISC-507 has one transmembrane domain (aa 671-687), a large N-terminalextracellular and a shorter C-terminal cytoplasmatic region.

ISC-507 expression has been reported to be specifically restricted tomammalian epididymis, the small gland adjacent to the testicle, which iscritically involved in maturation of sperm. According to literature,ISC-507 is transferred from the epididymis to the sperm surface andredistributed in the sperm head during acrosome reaction (Adachi et al.,Mol Reprod Dev. 64:414-21, 2003).

RT-PCR investigations with ISC-507 specific primers (SEQ ID NO:7, 8)confirmed selective expression in the testis and absence of ISC-507 fromany other normal tissue (tab.2, FIG. 3), except weak expression inprostate and lymph node derived tissues (tab.2, FIG. 4).

However and most surprisingly, we observed expression of ISC-507 in asignificant number of prostate cancers (FIG. 3,4). This protein had notbeen reported before to be involved in cancer.

TABLE 2 ISC-507 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma − Cerebellum −Pancreatic − carcinoma Myocardium − Esophageal − carcinoma Skeletalmuscle − Stomach carcinoma − Myocardium Lung cancer − Stomach − Breastcancer − Colon − Ovarian carcinoma − Pancreas − Uterus carcinoma −Kidney − Head & Neck Cancer − Liver − Kidney cancer − Testis +++Prostate carcinoma +++ Thymus − Liver carcinoma − Breast − Ovary −Uterus − Skin − Lung − Placenta − Lymph nodes + Spleen − PBMC − Prostate+

The absence from toxicity relevant normal tissues and the frequent andsignificant expression of ISC-507 in prostate cancers make this proteinaccording to the invention a valuable diagnostic and therapeutic marker.This includes according to the invention the detection of disseminatedtumor cells in serum, bone marrow, urine, and the detection ofmetastases in other organs by means of RT-PCR. In addition, theextracellular domains of ISC-507 can be used according to the inventionas target structure for immunodiagnosis and therapy by means ofmonoclonal antibodies. In addition, ISC-507 can be employed according tothe invention as vaccine (RNA, DNA, protein, peptides) for inducingtumor-specific immune responses (T and B cell-mediated immuneresponses).

Antibodies for detecting ISC-507 could be produced with followingpeptides and proteins: SEQ ID NO:51, 52, 53, 54, 55, 6, 56 and 57.

According to the invention an antibody which binds to ISC-507 might beuseful for therapeutic or diagnostic purposes.

Example 3 Identification of ISC-466 as Therapeutic and Diagnostic CancerTarget

ISC-466 (SEQ ID NO:9) encodes a 426 aa protein (SEQ ID NO:10) with amolecular weight of 48.2 kDA. It belongs to the family ofpregnancy-specific glycoproteins. The human pregnancy-specificglycoproteins (PSGs) are a group of molecules that are mainly producedby the placental syncytiotrophoblasts during pregnancy and are part ofthe immunoglobulin superfamily (Beauchemin et al., Exp Cell Res.252(2):243-9, 1999)

As other PSGs, ISC-466 as well has been reported to be restricted toplacenta.

According to the invention, a gene-specific primer pair (SEQ ID NO:11,12) for ISC-466 was used in RT-PCR analyses to amplify cDNA derived froma comprehensive panel of normal and tumor tissues. The RT-PCR analysisreveals expression of ISC-466 transcripts in normal placenta, and weakexpression in thymus and ovary (tab.3, FIG. 5A). No significantexpression was detected in any other normal organ tissue. Mostsurprisingly, when cancer cell lines were investigated, we found highand significant levels of expression in a number of tumor types,including breast cancer (FIG. 5C), lung cancer (FIG. 5C), ovariancarcinoma (FIG. 5D) and head and neck- and kidney carcinomas (FIG. 5B).

TABLE 3 ISC-466 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma + Thymus +Pancreatic + carcinoma Myocardium − Esophageal + carcinoma Skeletalmuscle − Stomach carcinoma − Myocardium Lung cancer ++ Stomach − Breastcancer +++ Colon − Ovarian carcinoma ++ Pancreas − Cervix carcinoma −Kidney − Head & Neck Cancer +++ Liver − Kidney cancer ++ Testis +++Prostate carcinoma + Thymus − Liver carcinoma − Breast − Melanoma +Ovary + Uterus − Skin − Lung − Placenta − Lymph nodes − Spleen − PBMC −Prostate −

In contrast to the observation, that ISC-466 is involved by colorectalcarcinomas (Salahshor et al., BMC Cancer. 5:66, 2005), ourinvestigations reveal ISC-466 according to the invention as diagnosticand therapeutic marker for head & neck, breast, ovarian, prostate cancerand melanoma.

Example 4 Identification of ISC-518 as Therapeutic and Diagnostic CancerTarget

ISC-518 (SEQ ID NO:13) encodes a 237 aa translation product (SEQ IDNO:14). However, no data with regard to tissue distribution and noconnection to cancer is available so far.

ISC-518 is a hypothetical, bioinformaticly predicted gene/protein.Sequence analyses revealed that the protein has a transmembrane domain(aa 102-118). The extracellular C-terminus features a functional domain,which occurs in cell-surface glycoproteins.

According to the invention, a gene-specific primer pair (SEQ ID NO:15,16) for ISC-518 was used in RT-PCR analyses to amplify cDNA derived froma comprehensive panel of normal and tumor tissues. The only normaltissue we found to express this gene was testis, whereas no significantexpression of ISC-518 was detectable in any other normal organ (FIG. 6).Most surprisingly, when cancer specimen were investigated, we found highand significant levels of expression in hepatocarcinomas (FIG. 7)

TABLE 4 ISC-518 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma − Cerebellum −Pancreatic − carcinoma Myocardium − Esophageal − carcinoma Skeletalmuscle − Stomach carcinoma + Myocardium Lung cancer + Stomach − Breastcancer + Colon − Ovarian carcinoma + Pancreas − Uterus carcinoma −Kidney − Head & Neck Cancer − Liver − Kidney cancer − Testis +++Prostate carcinoma + Thymus − Liver carcinoma ++ Breast − Ovary − Uterus− Skin − Lung − Placenta − Lymph nodes − Spleen − PBMC − Prostate −

Bioinformatic investigations showed that the protein encoded by ISC-518represents a cell surface molecule. The previous unknown selectiveexpression of this surface molecule makes it a target for therapeuticpurposes and for developing diagnostic methods for the detection oftumor cells and therapeutic methods for the elimination of tumor cells.

Example 5 Identification of ISC-477 as Therapeutic and Diagnostic CancerTarget

ISC-477 (SEQ ID NO:17) encodes a 130 aa translation product (SEQ IDNO:18). ISC-477 is a hypothetical protein. No data with regard to tissuedistribution and no connection to cancer was publicly available.

Structural analysis reveals a hydrophobic region, which might be atransmembrane region or signal peptide.

According to the invention, a gene-specific primer pair (SEQ ID NO: 19,20) for ISC-477 was used in RT-PCR analyses to amplify cDNA derived froma comprehensive panel of normal and tumor tissues. The only normaltissues we found to express this gene were placenta and ovary. Incontrast, no significant expression of ISC-477 was detectable in anyother normal organ (FIG. 8A). Most surprisingly, when cancer specimenswere investigated, we found high and significant levels of expression inlung, ovarian, colon and stomach cancer (FIG. 8A-D). Expression levelsare clearly higher than expression in normal ovary.

TABLE 5 ISC-477 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma ++ Cerebellum −Pancreatic + carcinoma Myocardium − Esophageal − carcinoma Skeletalmuscle − Stomach carcinoma ++ Myocardium Lung cancer +++ Stomach −Breast cancer ++ Colon − Ovarian carcinoma ++ Pancreas − Kidney cancer −Kidney − Prostate carcinoma − Liver − Liver carcinoma − Testis − Thymus− Breast − Ovary ++ Uterus − Skin − Lung − Placenta +++ Lymph nodes −Spleen − PBMC − Prostate −

Example 6 Identification of ISC-489 as Therapeutic and Diagnostic CancerTarget

ISC-489 (SEQ ID NO:21) encodes a 363 aa translation product (SEQ IDNO:22). The protein is a newly described member of the family ofG-protein coupled receptors. However, no data with regard to tissuedistribution and no connection to cancer was publically available.

According to the invention, a gene-specific primer pair (SEQ IDNO:23,24) for ISC-489 was used in RT-PCR analyses to amplify cDNAderived from a comprehensive panel of normal and tumor tissues. The onlynormal tissues we found to express this gene were placenta and esophagus(weak expression). In contrast, no significant expression of ISC-489 wasdetectable in any other normal organ (FIG. 9A). Most surprisingly, whencancer specimens were investigated, we found high and significant levelsof expression in head and neck, and stomach cancers (FIG. 9B, 9C).

As member of the G-protein coupled receptor family, ISC-489 is anintegral membrane protein with 7 transmembrane domains and severalextracellular loops, which can be targeted on the cell surface.

TABLE 6 ISC-489 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma + Esophagus +Stomach carcinoma ++ Myocardium − Lung cancer + Skeletal muscle − Breastcancer − Myocardium Ovarian carcinoma − Stomach − Head & Neck Cancer +++Colon − Kidney cancer + Pancreas − Prostate carcinoma − Kidney − Livercarcinoma + Liver − Testis − Thymus − Breast − Ovary − Uterus − Skin −Lung − Placenta +++ Lymph nodes − Spleen − PBMC − Prostate −

The pronounced expression and unexpected high incidence of ISC-489 inhead and neck carcinomas make this protein according to the invention ahighly interesting diagnostic and therapeutic marker.

Example 7 Identification of ISC-461 as Therapeutic and Diagnostic CancerTarget

ISC-461 (SEQ ID NO:25) encodes a 419 aa protein (SEQ ID NO:26) with amolecular weight of 47.1 kDA.

It belongs to the family of pregnancy-specific glycoproteins. The humanpregnancy-specific glycoproteins (PSGs) are a group of molecules thatare mainly produced by the placental syncytiotrophoblasts duringpregnancy and are part of the immunoglobulin superfamily (Beauchemin etal., Exp Cell Res. 252(2):243-9, 1999).

As other PSGs, ISC-461 as well has been reported to be restricted toplacenta.

According to the invention, a gene-specific primer pair (SEQ ID NO:11,27) for ISC-461 was used in RT-PCR analyses to amplify cDNA derived froma comprehensive panel of normal and tumor tissues. As expected, placentawas confirmed as expressing this gene, besides weak expression in testisand ovary (FIGS. 10A and 10B). No significant expression, whatsoever,was detected in any other normal organ tissue. Most surprisingly, whencancer derived tissues and cancer cell lines were investigated, we foundhigh and significant levels of expression in a number of tumor types,including breast cancer (FIG. 10C), ovarian carcinoma (FIG. 10D) andmelanoma (FIG. 10B, 10C).

TABLE 7 ISC-461 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma − Cerebellum −Pancreatic + carcinoma Myocardium − Esophageal + carcinoma Skeletalmuscle − Stomach carcinoma + Myocardium Lung cancer + Stomach − Breastcancer ++ Colon − Ovarian carcinoma ++ Pancreas − Kidney cancer − Kidney− Prostate carcinoma − Liver − Liver carcinoma − Testis + Melanoma ++Thymus − Breast − Ovary + Uterus − Skin − Lung − Placenta +++ Lymphnodes − Spleen − PBMC − Prostate −

A further aim according to the invention was to identify splice variantsfor ISC-461 which can be utilized both for diagnosis and for therapy.

On investigation of splice variants we could identify a splice form (SEQID NO:28) and the protein encoded thereby (SEQ ID NO:29).

Example 8 Identification of ISC-465 as Therapeutic and Diagnostic CancerTarget

ISC-465 (SEQ ID NO:30) encodes a 419 aa protein (SEQ ID NO:31) with amolecular weight of 47.0 kDA.

It belongs to the family of pregnancy-specific glycoproteins. The humanpregnancy-specific glycoproteins (PSGs) are a group of molecules thatare mainly produced by the placental syncytiotrophoblasts duringpregnancy and are part of the immunoglobulin superfamily (Beauchemin etal., Exp Cell Res. 252(2):243-9, 1999).

As other PSGs, ISC-465 as well has been reported to be restricted toplacenta.

According to the invention, a gene-specific primer pair (SEQ ID NO:11,32) for ISC-465 was used in RT-PCR analyses to amplify cDNA derived froma comprehensive panel of normal and tumor tissues. As expected, placentawas confirmed as expressing this gene, besides weak expression in normalovary (FIG. 11A). No significant expression, whatsoever, was detected inany other normal organ tissue. Most surprisingly, when cancer derivedtissues and cancer cell lines were investigated, we found high andsignificant levels of expression in a number of tumor types (FIG. 11A,11B), especially breast cancer (FIG. 11B).

TABLE 8 ISC-461 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma − Cerebellum −Pancreatic + carcinoma Myocardium − Esophageal + carcinoma Skeletalmuscle − Stomach carcinoma + Myocardium Lung cancer + Stomach − Breastcancer ++ Colon − Ovarian carcinoma ++ Pancreas − Kidney cancer − Kidney− Prostate carcinoma − Liver − Liver carcinoma − Testis + Melanoma +Thymus − Breast − Ovary + Uterus − Skin − Lung − Placenta +++ Lymphnodes − Spleen − PBMC − Prostate −

The selective and high expression of ISC-465 transcripts in tumors wasnot previously known and can be utilized according to the invention formolecular diagnostic methods such as RT-PCR for detecting disseminatingtumor cells in the serum and bone marrow and for detecting metastases inother tissues. This molecule can be further used as specific target fortherapeutic approaches.

Example 9 Identification of Mem-030 as Therapeutic and Diagnostic CancerTarget

Mem-030 (SEQ ID NO:35) encodes a 592 aa protein (SEQ ID NO:36) with amolecular weight of 67.9 kDA.

Mem-030 belongs to the GBP-proteins, which are large GTPases being ableto bind GTP, GDP, and GMP and to catalyze the hydrolysis of GTP to GDP,as well as GMP (Cheng et al., J. Biol. Chem. 260:15834-9, 1985). GTPasesplay an important role in cell proliferation, differentiation, signaltransduction, and intracellular protein transportation and areinterferon inducible (Boehm et al., J. Immunol. 161(12):6715-23, 1998).

Also, Mem-030 counteracts the proliferative effect of inflammatorycytokines like IFN-g, interleukin 1-b (IL-1b), and tumor necrosisfactor-a (TNF-a) 1 on endothelial cells (Guenzi et al., EMBO J.20(20):5568-77, 2001).

According to the invention, a gene-specific primer pair (SEQ ID NO:37,38) for Mem-030 was used in real time RT-PCR analyses to amplify cDNAderived from a comprehensive panel of normal and tumor tissues. Mem-030show an ubiquitous expression pattern (FIG. 12A, tab.9).

Most surprisingly, when cancer derived tissues and cancer cell lineswere investigated, we found high and significant levels ofoverexpression in a number of tumor types (FIG. 12A, 12B), especiallyhead and neck carcinomas.

TABLE 9 Mem-030 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain + Colon carcinoma + Myocardium +Pancreatic + carcinoma Skeletal muscle + Esophageal ++ carcinomaMyocardium + Stomach carcinoma + Stomach + Lung cancer + Colon + Breastcancer + Pancreas + Ovarian carcinoma + Kidney + Uterus carcinoma ++Liver + Head & Neck Cancer +++ Testis + Kidney cancer + Thymus +Prostate carcinoma + Breast + Liver carcinoma ++ Ovary + Melanoma ++Uterus + Skin + Lung + Placenta + Lymph nodes + Spleen + PBMC + Prostate+

Due to bioinformatics and literature analysis, a homologous gene ofMem-030 might be also an attractive therapeutic target (SEQ ID NO:39)and encodes a 586 aa protein (SEQ ID NO:40) with a molecular weight of66.6 kDA.

Bioinformatic investigations showed that both proteins represent cellsurface molecules. The previously unknown selective overexpression ofthis surface molecule makes it a target for therapeutic purposes and fordeveloping diagnostic methods for the detection of tumor cells andtherapeutic methods for the elimination of tumor cells.

Example 10 Identification of Mem-055 as Therapeutic and DiagnosticCancer Target

Mem-055 (SEQ ID NO:41) encodes a 250 aa protein (SEQ ID NO:42) with amolecular weight of 27.9 kDA.

The protein encoded by this gene is a lysosomal thiol reductase that atlow pH can reduce protein disulfide bonds. The enzyme is expressedconstitutively in antigen-presenting cells and induced bygamma-interferon in other cell types. This enzyme has an important rolein MHC class II-restricted antigen processing (Arunachalam et al. ProcNatl Acad Sci USA. 97(2):745-50, 2000).

The localization of Mem-055 and the protein topology was predicted byanalysis of the putative signal sequences and transmembrane domains withbioinformatic tools (TMPRED, SOUSI). Mem-055 might have an extracellularC-terminus.

According to the invention, a gene-specific primer pair (SEQ ID NO:43,44) for Mem-055 was used in real time RT-PCR analyses to amplify cDNAderived from a comprehensive panel of normal and tumor tissues. Mem-055show an ubiquitous expression pattern (FIG. 13A, tab.10).

Most surprisingly, when Mem-055 expression within cancer derived tissueswas investigated, we found high and significant levels of overexpressionin a number of tumor types (FIG. 13A, 13B), especially stomach cancers.

TABLE 10 Mem-055 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain + Colon carcinoma + Myocardium +Pancreatic + carcinoma Skeletal muscle + Esophageal + carcinomaMyocardium + Stomach carcinoma +++ Stomach + Lung cancer ++ Colon +Breast cancer ++ Pancreas + Ovarian carcinoma ++ Kidney + Uteruscarcinoma + Liver + Head & Neck Cancer + Testis + Kidney cancer +Thymus + Prostate carcinoma + Breast + Liver carcinoma ++ Ovary +Melanoma + Uterus + Skin + Lung + Placenta + Lymph nodes + Spleen +PBMC + Prostate +

Mem-055 is a target structure for therapeutic and diagnostic purposes,because of the putative extracellular domain and the unexpectedoverexpression in different carcinoma types.

Example 11 Identification of Mem-062 as Therapeutic and DiagnosticCancer Target

Mem-062 (SEQ ID NO:45) encodes a 271 aa protein (SEQ ID NO:46) with amolecular weight of 30.7 kDA.

By a computer-based screening method Mem-062 could previously beidentified and was described as testis, prostate and placentaspecifically expressed (Bera et al., Biochem Biophys Res Commun.312(4):1209-15, 2003)

According to the invention, a gene-specific primer pair (SEQ ID NO:47,48) for Mem-062 was used in RT-PCR analyses. Mem-062 surprisingly showeda cancer-testis specific expression pattern (FIG. 14A, tab.11). Noexpression was detected in any other normal organ tissue. Mostsurprisingly, when cancer derived tissues were investigated, we foundsignificant levels of Mem-62 expression (FIG. 14B), especially inovarian carcinomas.

TABLE 11 Mem-062 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma + Myocardium −Pancreatic − carcinoma Skeletal muscle − Esophageal − carcinomaMyocardium − Stomach carcinoma − Stomach − Lung cancer − Colon − Breastcancer − Pancreas − Ovarian carcinoma ++ Kidney − Uterus carcinoma −Liver − Head & Neck Cancer − Testis − Kidney cancer − Thymus − Prostatecarcinoma − Breast − Liver carcinoma − Ovary − Melanoma − Uterus − Skin− Lung − Placenta − Lymph nodes − Spleen − PBMC − Prostate −

Alternative splicing results in an alternative transcript (SEQ ID NO:49)and its corresponding translation product (SEQ ID NO:50).

Example 12 Identification of Mem-068 as Therapeutic and DiagnosticCancer Target

Mem-068 (SEQ ID NO:61) is a newly identified cDNA clone.

By a bioinformatic prediction approach (Genscan) Mem-068 could bedescribed as multiple exon gene on chromosome 9 (SEQ ID NO:62). Thededuced protein sequence (SEQ ID NO:63) has 751 aa and forms a proteinwith a molecular weight of 82.4 kDA.

According to the invention, a gene-specific primer pair for Mem-068 wasused in RT-PCR analyses. Mem-068 show surprisingly a cancer-testisspecific expression pattern (FIG. 15A, tab.12). No expression wasdetected in any other normal organ tissue except placenta (weakexpression). Most surprisingly, when cancer derived tissues wereinvestigated, we found significant levels of Mem-068 expressed (FIG.15B), especially in renal cell carcinomas and in stomach cancers.

TABLE 12 Mem-068 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma + Breast Renalcell carcinoma ++ Colon − Stomach carcinoma + Kidney − Lung cancer +Liver − Breast cancer − Lung − Ovarian carcinoma − Lymph nodes −Melanoma − Ovary − Prostate carcinoma − Pancreas − Placenta + PBMC −PBMC activated − Prostate − Skeletal muscle − Skin − Stomach − Spleen −Testis + Uterus −

According to the transmembrane prediction programme TMpred Mem-068 mightbe expressed at the cell surface, which makes it an interesting targetfor therapeutic or diagnostic purposes.

Example 13 Identification of Mem-071 as Therapeutic and DiagnosticCancer Target

Mem-071 (SEQ ID NO:64) is a new cDNA clone, which is encoded in 2 exonson chromosome 1.

According to the invention, a gene-specific primer pair for Mem-071 wasused in RT-PCR analyses to amplify cDNA derived from a comprehensivepanel of normal and tumor tissues. The only normal tissues we found toexpress this gene was testis (FIG. 16A). In contrast, when cancerspecimen were investigated, we found high and significant levels ofexpression in renal cell carcinomas and stomach cancers (FIG. 16B).

TABLE 13 Mem-071 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma − Breast −Renal cell carcinoma ++ Colon − Stomach carcinoma + Kidney − Lung cancer− Liver − Breast cancer − Lung − Ovarian carcinoma − Lymph nodes −Melanoma − Ovary − Prostate carcinoma − Pancreas − Placenta − PBMC −PBMC activated − Prostate − Skeletal muscle − Skin − Stomach − Spleen −Testis + Uterus −

The unexpected high incidence of Mem-071 in renal cell carcinomas makethis protein according to the invention a highly interesting diagnosticand therapeutic marker.

Example 14 Identification of Mem-072 as Therapeutic and DiagnosticCancer Target

Mem-072 (SEQ ID NO:65) is a newly identified gene, which is encoded in 3exons on chromosome 16.

According to the invention, a gene-specific primer pair for Mem-072 wasused in RT-PCR analyses to amplify cDNA derived from a comprehensivepanel of normal and tumor tissues. No expression within all testednormal tissues could be found (FIG. 17A, tab.14). When cancer derivedtissues and cancer cell lines were investigated, we found high andsignificant levels of expression in lung cancer samples (FIG. 17B).

TABLE 14 Mem-072 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma − Breast −Renal cell carcinoma − Colon − Stomach carcinoma − Kidney − Lung cancer++ Liver − Breast cancer − Lung − Ovarian carcinoma − Lymph nodes −Melanoma − Ovary − Prostate carcinoma − Pancreas − Placenta − PBMC −PBMC activated − Prostate − Skeletal muscle − Skin − Stomach − Spleen −Testis − Uterus −

The selective and high expression of Mem-072 in lung tumors was notpreviously known and can be utilized according to the invention formolecular diagnostic methods such as RT-PCR for detecting disseminatingtumor cells in the serum and bone marrow and for detecting metastases inother tissues. This molecule can be further used as specific target fortherapeutic approaches.

Example 15 Identification of Mem-106 as Therapeutic and DiagnosticCancer Target

Mem-106 (SEQ ID NO:66) is a newly identified cDNA, which is intronlessencoded on chromosome 2.

According to the invention, a gene-specific primer pair for Mem-106 wasused in RT-PCR analyses. Mem-106 surprisingly showed a cancer-testisspecific expression pattern (FIG. 18A, tab.15). No expression wasdetected in any other normal organ tissue. Most surprisingly, whencancer derived tissues were investigated, we found significant levels ofMem-106 expression (FIG. 18B), especially in ovarian carcinomas.

TABLE 15 Mem-106 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Breast − Colon carcinoma + Colon −Renal cell carcinoma − Kidney − Stomach carcinoma − Liver − Lung cancer− Lung − Breast cancer − Lymph nodes − Ovarian carcinoma ++ Ovary −Melanoma ++ Pancreas − Prostate carcinoma − Placenta − PBMC − PBMCactivated − Prostate − Skeletal muscle − Skin − Stomach − Spleen −Testis ++ Uterus −

Mem-106 is a target structure for therapeutic and diagnostic purposes,because of the unexpected overexpression in different carcinoma types.

Example 16 Identification of Mem-131 as Therapeutic and DiagnosticCancer Target

Mem-131 (SEQ ID NO:67) is a newly identified cDNA clone. Mem-131 is a 2exon gene on chromosome 15.

According to the invention, a gene-specific primer pair for Mem-131 wasused in RT-PCR analyses to amplify cDNA derived from a comprehensivepanel of normal and tumor tissues. The RT-PCR analysis revealsexpression of Mem-131 transcripts only in normal activated PBMCs(tab.16, FIG. 19A). No significant expression was detected in any othernormal organ tissue. Most surprisingly, when cancer samples wereinvestigated, we found high and significant levels of expression in anumber of tumor types, including breast cancer (FIG. 19B), lung cancer(FIG. 19B+C) and ovarian carcinoma (FIG. 19C).

TABLE 16 Mem-131 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Breast − Lung cancer ++ Duodenum −Breast cancer ++ Bladder − Ovarian carcinoma ++ Skin − Brain − Bonemarrow − Colon − Liver − Lung − Lymph node − Stomach − Spleen − Myocard− Kidney − Esophagus − Ovary − Pancreas − PBMC − PBMC activated ++Placenta − Muscle − Testis − Thymus −

Our investigations reveals Mem-131 according to the invention asdiagnostic and therapeutic marker for lung, breast and ovarian cancers.

1.-6. (canceled)
 7. A pharmaceutical composition, comprising one or morecomponents selected from the group consisting of: (i) a tumor-associatedantigen or a part thereof, (ii) a nucleic acid which codes for atumor-associated antigen or a part thereof, (iii) an antibody whichbinds to a tumor-associated antigen or a part thereof, (iv) an antisensenucleic acid which hybridizes specifically with a nucleic acid codingfor a tumor-associated antigen, (v) an siRNA directed against a nucleicacid coding for a tumor-associated antigen, (vi) a host cell whichexpresses a tumor-associated antigen or a part thereof, and (vii)isolated complexes between a tumor-associated antigen or a part thereofand an MHC molecule, said tumor-associated antigen having a sequenceencoded by a nucleic acid which is selected from the group consistingof: (a) a nucleic acid which comprises a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 5, 1, 9, 13, 17, 21, 25, 28,30, 35, 39, 41, 45, 49, 61, 62, and 64-67, a part or derivative thereof,(b) a nucleic acid which hybridizes with the nucleic acid of (a) understringent conditions, (c) a nucleic acid which is degenerate withrespect to the nucleic acid of (a) or (b), and (d) a nucleic acid whichis complementary to the nucleic acid of (a), (b) or (c).
 8. (canceled)9. (canceled)
 10. The pharmaceutical composition as claimed in claim 7,in which the host cell additionally expresses an MHC molecule whichbinds to the tumor-associated antigen or the part thereof. 11.(canceled)
 12. (canceled)
 13. The pharmaceutical composition as claimedin claim 7, in which the host cell is an antigen-presenting cell. 14.The pharmaceutical composition as claimed in claim 7, in which theantibody is a monoclonal, chimeric or humanized antibody, or is afragment of an antibody.
 15. The pharmaceutical composition as claimedin claim 7, in which the antibody is coupled to a therapeutic ordiagnostic agent.
 16. The pharmaceutical composition as claimed in claim7, for use in the treatment or prevention of cancer.
 17. Thepharmaceutical composition as claimed in claim 16, in which the canceris a lung tumor, a breast tumor, a prostate tumor, a melanoma, a colontumor, a gastric tumor, a pancreatic tumor, an ENT tumor, a renal cellcarcinoma or a cervical carcinoma, a colon carcinoma or a mammarycarcinoma.
 18. The pharmaceutical composition as claimed in claim 7, inwhich the tumor-associated antigen comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 6, 51-57, 2, 10, 14,18, 22, 26, 29, 31, 36, 40, 42, 46, 50, 58-60, 63, 68, and 69, a partthereof, or derivative thereof. 19.-30. (canceled)
 31. A method oftreating or preventing a disease characterized by expression or abnormalexpression of a tumor-associated antigen, which method comprises thestep of administration of a pharmaceutical composition as claimed inclaim 7, said tumor-associated antigen having a sequence encoded by anucleic acid which is selected from the group consisting of: (a) anucleic acid which comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 5, 1, 9, 13, 17, 21, 25, 28, 30, 35, 39,41, 45, 49, 61, 62, and 64-67, a part or derivative thereof, (b) anucleic acid which hybridizes with the nucleic acid of (a) understringent conditions, (c) a nucleic acid which is degenerate withrespect to the nucleic acid of (a) or (b), and (d) a nucleic acid whichis complementary to the nucleic acid of (a), (b) or (c).
 32. (canceled)33. (canceled)
 34. A method of treating a patient having a diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen, which method comprises the steps of: (i) providing a samplecontaining immunoreactive cells, (ii) contacting said sample with a hostcell expressing said tumor-associated antigen or a part thereof, underconditions which favor production of cytolytic or cytokine-releasing Tcells against said tumor-associated antigen or said part thereof, and(iii) introducing the cytolytic or cytokine-releasing T cells into thepatient in an amount suitable for lysing cells expressing thetumor-associated antigen or a part thereof, said tumor-associatedantigen having a sequence encoded by a nucleic acid which is selectedfrom the group consisting of: (a) a nucleic acid which comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:5, 1, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67,a part or derivative thereof, (b) a nucleic acid which hybridizes withthe nucleic acid of (a) under stringent conditions, (c) a nucleic acidwhich is degenerate with respect to the nucleic acid of (a) or (b), and(d) a nucleic acid which is complementary to the nucleic acid of (a),(b) or (c).
 35. The method as claimed in claim 34, in which-the hostcell recombinantly or endogenously expresses an MHC molecule binding tothe tumor-associated antigen or to a part thereof.
 36. (canceled) 37.(canceled)
 38. The method as claimed in claim 31, in which thetumor-associated antigen comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 6, 51-57, 2, 10, 14, 18, 22, 26, 29,31, 36, 40, 42, 46, 50, 58-60, 63, 68, and 69, a part or derivativethereof.
 39. An antibody, which binds specifically to a protein orpolypeptide or to a part thereof, said protein or polypeptide beingencoded by a nucleic acid selected from the group consisting of: (a) anucleic acid which comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 5, 1, 9, 13, 17, 21, 25, 28, 30, 35, 39,41, 45, 49, 61, 62, and 64-67, a part or derivative thereof, (b) anucleic acid which hybridizes with the nucleic acid of (a) understringent conditions, (c) a nucleic acid which is degenerate withrespect to the nucleic acid of (a) or (b), and (d) a nucleic acid whichis complementary to the nucleic acid of (a), (b) or (c).
 40. Theantibody as claimed in claim 39, in which the protein or polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 6, 51-57, 2, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50,58-60, 63, 68, and 69, and a part or derivative thereof.
 41. (canceled)42. The antibody as claimed in claim 39, in which the antibody is amonoclonal, chimeric or humanized antibody, or is a fragment of anantibody.
 43. (canceled)
 44. (canceled)
 45. (canceled)
 46. A conjugatebetween an antibody as claimed in claim 39 and a therapeutic ordiagnostic agent.
 47. The conjugate as claimed in claim 46, in which thetherapeutic or diagnostic agent is a toxin.
 48. (canceled)
 49. Themethod as claimed in claim 34, in which the tumor-associated antigencomprises an amino acid sequence selected from the group consisting of:SEQ ID NOs: 6, 51-57, 2, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50,58, 59, 60, 63, 68, and 69, and a part or derivative thereof.